Reaction of ammonium triphosphate with gadolinium nitrate in aqueous solution at 273K

International Nuclear Information System (INIS)

Rodicheva, G.V.; Tananaev, I.V.; Romanova, N.M.

1982-01-01

The solubility in the system (NW 4 ) 5 P 3 O 10 -Gd(NO 3 ) 3 - H 2 O (273 K) is studied. Depending on the reagent ratio formation of the compounds Gd 5 (P 3 O 10 ) 3 x22H 2 O, NH 4 Gd 3 (P 3 O 10 ) 2 x12H 2 O and (NH 4 ) 3 Gd 4 (P 3 O 10 ) 3 x14H 2 O is established. Gadolinium triphosphates, separated from solution, are studied using the methods of paper chromatography, X-ray diffractometry, thermography. Simultaneously with thermal dehydration of gadolinium triphosphates the processes of triphosphate decomposition and phosphate anion condensation take place. A mixture of crystalline ortho-phosphate and long- chain polyphosphate of gadolinium is the final product of thermal decomposition (1063 K) of normal and doubl e ammonium- containing gadolinium triphosphates [ru

A terbium(III)-organic framework for highly selective sensing of cytidine triphosphate.

Science.gov (United States)

Zhao, Xi Juan; He, Rong Xing; Li, Yuan Fang

2012-11-21

Highly selective sensing of cytidine triphosphate (CTP) against other triphosphate nucleosides including ATP, GTP and UTP is successfully achieved with a luminescent terbium(III)-organic framework (TbOF) of [Tb(2)(2,3-pzdc)(2)(ox)(H(2)O)(2)](n) (2,3-pzdc(2-) = 2,3-pyrazinedicarboxylate, ox(2-) = oxalate).

Piracetam prevents scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities.

Science.gov (United States)

Marisco, Patricia C; Carvalho, Fabiano B; Rosa, Michelle M; Girardi, Bruna A; Gutierres, Jessié M; Jaques, Jeandre A S; Salla, Ana P S; Pimentel, Víctor C; Schetinger, Maria Rosa C; Leal, Daniela B R; Mello, Carlos F; Rubin, Maribel A

2013-08-01

Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.

17β-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes.

Science.gov (United States)

Mitrović, Nataša; Zarić, Marina; Drakulić, Dunja; Martinović, Jelena; Sévigny, Jean; Stanojlović, Miloš; Nedeljković, Nadežda; Grković, Ivana

2017-03-01

17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24 h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.

Oral sucrose for heel lance enhances adenosine triphosphate use in preterm neonates with respiratory distress.

Science.gov (United States)

Angeles, Danilyn M; Asmerom, Yayesh; Boskovic, Danilo S; Slater, Laurel; Bacot-Carter, Sharon; Bahjri, Khaled; Mukasa, Joseph; Holden, Megan; Fayard, Elba

2015-01-01

To examine the effects of oral sucrose on procedural pain, and on biochemical markers of adenosine triphosphate utilization and oxidative stress in preterm neonates with mild to moderate respiratory distress. Preterm neonates with a clinically required heel lance that met study criteria (n = 49) were randomized into three groups: (1) control (n = 24), (2) heel lance treated with placebo and non-nutritive sucking (n = 15) and (3) heel lance treated with sucrose and non-nutritive sucking (n = 10). Plasma markers of adenosine triphosphate degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile. Data were analyzed using repeated measures analysis of variance, chi-square and one-way analysis of variance. We found that in preterm neonates who were intubated and/or were receiving ⩾30% FiO2, a single dose of oral sucrose given before a heel lance significantly increased markers of adenosine triphosphate use. We found that oral sucrose enhanced adenosine triphosphate use in neonates who were intubated and/or were receiving ⩾30% FiO2. Although oral sucrose decreased pain scores, our data suggest that it also increased energy use as evidenced by increased plasma markers of adenosine triphosphate utilization. These effects of sucrose, specifically the fructose component, on adenosine triphosphate metabolism warrant further investigation.

Rat pancreas secretes particulate ecto-nucleotidase CD39

DEFF Research Database (Denmark)

Sørensen, Christiane Elisabeth; Amstrup, Jan; Rasmussen, Hans N

2003-01-01

In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar......-ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization...... relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK-8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti-CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic...

Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

Science.gov (United States)

Zhang, Zhanxia; Sharon, Etery; Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

2012-06-05

The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 μM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

Stability of [5-3H]uridine-5'-triphosphate

International Nuclear Information System (INIS)

Brabec, D.

1980-01-01

The effect of temperature, the solvent systems, evaporation, volume reduction, etc. on the decomposition rate of [5- 3 H]uridine-5'-triphosphate was investigated. The decomposition rates and optimum storage conditions were established. The possibility of reducing the duration of the purification and separation process was examined. (author)

21 CFR 864.7040 - Adenosine triphosphate release assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7040...

New crystal forms of NTPDase1 from the bacterium Legionella pneumophila

International Nuclear Information System (INIS)

Zebisch, Matthias; Schäfer, Petra; Lauble, Peter; Sträter, Norbert

2013-01-01

The soluble NTPDase1 from L. pneumophila was crystallized in six crystal forms and the structure was solved using a sulfur SAD approach. Nucleoside triphosphate diphosphohydrolases (NTPDases) are a large class of nucleotidases that hydrolyze the (γ/β)- and (β/α)-anhydride bonds of nucleoside triphosphates and diphosphates, respectively. NTPDases are found throughout the eukaryotic domain. In addition, a very small number of members can be found in bacteria, most of which live as parasites of eukaryotic hosts. NTPDases of intracellular and extracellular parasites are emerging as important regulators for the survival of the parasite. To deepen the knowledge of the structure and function of this enzyme class, recombinant production of the NTPDase1 from the bacterium Legionella pneumophila has been established. The protein could be crystallized in six crystal forms, of which one has been described previously. The crystals diffracted to resolutions of between 1.4 and 2.5 Å. Experimental phases determined by a sulfur SAD experiment using an orthorhombic crystal form produced an interpretable electron-density map

The immediate nucleotide precursor, guanosine triphosphate, in the riboflavin biosynthetic pathway

International Nuclear Information System (INIS)

Mitsuda, Hisateru; Nakajima, Kenji; Nadamoto, Tomonori

1977-01-01

In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashbyii. The added purines, at 10 -4 M, were effectively incorporated into riboflavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the course of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed. (auth.)

Purinergic signaling modulates the cerebral inflammatory response in experimentally infected fish with Streptococcus agalactiae: an attempt to improve the immune response.

Science.gov (United States)

Souza, Carine F; Baldissera, Matheus D; Bottari, Nathiele B; Moreira, Karen L S; da Rocha, Maria Izabel U M; da Veiga, Marcelo L; Santos, Roberto C V; Baldisserotto, Bernardo

2018-06-01

Appropriate control of the immune response is a critical determinant of fish health, and the purinergic cascade has an important role in the immune and inflammatory responses. This cascade regulates the levels of adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate and adenosine (Ado), molecules involved in physiological or pathological events as inflammatory and anti-inflammatory mediators. Thus, the aim of this study was to evaluate whether purinergic signaling, through the activities of nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and adenosine deaminase (ADA), is capable of modulating the cerebral immune and inflammatory responses in silver catfish that is experimentally infected with Streptococcus agalactiae. Cerebral NTPDase (with ATP as substrate) and 5'-nucleotidase activities increased, while ADA activity decreased in silver catfish that is experimentally infected with S. agalactiae, compared to the control group. Moreover, the cerebral levels of ATP and Ado increased in infected animals compared to the uninfected control group. Brain histopathology in infected animals revealed inflammatory demyelination (the presence of occasional bubbly collections), increased cellular density in the area near to pia-mater and intercellular edema. Based on this evidence, the modulation of the purinergic cascade by the enzymes NTPDase, 5'-nucleotidase, and ADA exerts an anti-inflammatory profile due to the regulation of ATP and Ado levels. This suggests involvement of purinergic enzymes on streptococcosis pathogenesis, through regulating cerebral ATP and Ado levels, molecules known to participate in physiological or pathological events as inflammatory and anti-inflammatory mediators, respectively. In summary, the modulation of the cerebral purinergic cascade exerts an anti-inflammatory profile in an attempt to reduce inflammatory damage.

Determination of Zidovudine Triphosphate Intracellular Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Individuals by Tandem Mass Spectrometry

Science.gov (United States)

Font, Eva; Rosario, Osvaldo; Santana, Jorge; García, Hermes; Sommadossi, Jean-Pierre; Rodriguez, Jose F.

1999-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentrations of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP), are relatively low (low numbers of femtomoles per 106 cells) in HIV-infected patient peripheral blood mononuclear cells. Recently, several methods have used either high-performance liquid chromatography (HPLC) or solid-phase extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurements of ZDV-TP. The limit of detection (LOD) by these methods ranged from 20 to 200 fmol/106 cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HIV-infected patients using SPE and HPLC with tandem mass spectrometry for analysis. The LOD by this method is 4.0 fmol/106 cells with a linear concentration range of at least 4 orders of magnitude from 4.0 to 10,000 fmol/106 cells. In hispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h. Intracellular ZDV-TP concentrations in these patients ranged from 41 to 193 fmol/106 cells. The low LOD obtained with this method will provide the opportunity for further in vivo pharmacokinetic studies of intracellular ZDV-TP in different HIV-infected populations. Furthermore, this methodology could be used to perform simultaneous detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphate. PMID:10582890

Localization of RNA transcription sites in insect oocytes using microinjections of 5-bromouridine 5'-triphosphate.

Directory of Open Access Journals (Sweden)

Dmitry Bogolyubov

2007-06-01

Full Text Available In the present study we used 5-bromouridine 5'-triphosphate (BrUTP microinjections to localize the transcription sites in oocytes of insects with different types of the ovarium structure: panoistic, meroistic polytrophic, and meroistic telotrophic. We found that in an insect with panoistic ovaries (Acheta domesticus, oocyte nuclei maintain their transcription activity during the long period of oocyte growth. In insects with meroistic ovaries (Tenebrio molitor and Panorpa communis, early oocyte chromosomes were found to be transcriptionally active, and some transcription activity still persist while the karyosphere, a compact structure formed by all condensed oocyte chromosomes, begins to develop. At the latest stages of karyosphere development, no anti-Br-RNA signal was registered in the karyosphere.

Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

NARCIS (Netherlands)

Makarchikov, A.F.; Wins, P.; Janssen, E.E.W.; Wieringa, B.; Grisar, T.; Bettendorff, L.

2002-01-01

Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate

Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation.

Directory of Open Access Journals (Sweden)

Qian Feng

Full Text Available Upon viral infections, pattern recognition receptors (PRRs recognize pathogen-associated molecular patterns (PAMPs and stimulate an antiviral state associated with the production of type I interferons (IFNs and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5' triphosphate (5'ppp-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3, a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5'ppp. We show here that a 5'ppp-containing cloverleaf (CL RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

Science.gov (United States)

Grossmann, K; Friedrich, H; Seitz, U

1980-01-01

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I. PMID:7470092

NTPDase5/PCPH as a New Target in Highly Aggressive Tumors: A Systematic Review

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Paula Andreghetto Bracco

2014-01-01

Full Text Available The protooncogene PCPH was recently identified as being the ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5. This protooncogene is converted into an oncogene by a single base pair deletion, resulting in frame change and producing a premature stop codon, leading to a mutated protein (mt-PCPH with only 27 kDa, which is much smaller than the original 47 kDa protein. Overexpression of the PCPH as well as the mutated PCPH increases the cellular resistance to stress and apoptosis. This is intriguing considering that the active form, that is, the oncogene, is the mutated PCPH. Several studies analyzed the expression of NTPDase5/mt-PCPH in a wide range of tumor cells and evaluated its role and mechanisms in cancer and other pathogenic processes. The main point of this review is to integrate the findings and proposed theories about the role played by NTPDase5/mt-PCPH in cancer progression, considering that these proteins have been suggested as potential early diagnostic tools and therapy targets.

Inhibition of rat synaptic membrane Na⁺/K⁺-ATPase and ecto-nucleoside triphosphate diphosphohydrolases by 12-tungstosilicic and 12-tungstophosphoric acid.

Science.gov (United States)

Čolović, Mirjana B; Bajuk-Bogdanović, Danica V; Avramović, Nataša S; Holclajtner-Antunović, Ivanka D; Bošnjaković-Pavlović, Nada S; Vasić, Vesna M; Krstić, Danijela Z

2011-12-01

The in vitro influence of Keggin structure polyoxotungstates, 12-tungstosilicic acid, H(4)SiW(12)O(40) (WSiA) and 12-tungstophosphoric acid, H(3)PW(12)O(40) (WPA), and monomer Na(2)WO(4) × 2H(2)O on rat synaptic plasma membrane (SPM) Na(+)/K(+)-ATPase and E-NTPDase activity was studied, whereas the commercial porcine cerebral cortex Na(+)/K(+)-ATPase served as a reference. Dose-dependent Na(+)/K(+)-ATPase inhibition was obtained for all investigated compounds. Calculated IC(50) (10 min) values, in mol/l, for SPM/commercial Na(+)/K(+)-ATPase, were: 3.4 × 10(-6)/4.3 × 10(-6), 2.9 × 10(-6)/3.1 × 10(-6) and 1.3 × 10(-3)/1.5 × 10(-3) for WSiA, WPA and Na(2)WO(4) × 2H(2)O, respectively. In the case of E-NTPDase, increasing concentrations of WSiA and WPA induced its activity reduction, while Na(2)WO(4) × 2H(2)O did not noticeably affect the enzyme activity at all investigated concentrations (up to 1 × 10(-3)mol/l). IC(50) (10 min) values, obtained from the inhibition curves, were (in mol/l): 4.1 × 10(-6) for WSiA and 1.6 × 10(-6) for WPA. Monolacunary Keggin anion was found as the main active molecular species present under physiological conditions (in the enzyme assays, pH 7.4), for the both polyoxotungstates solutions (1 mmol/l), using Fourier transform infrared (FT-IR) and micro-Raman spectroscopy. Additionally, commercial porcine cerebral cortex Na(+)/K(+)-ATPase was exposed to the mixture of Na(2)WO(4) × 2H(2)O and WSiA at different concentrations. Additive inhibition effect was achieved for lower concentrations of Na(2)WO(4) × 2H(2)O/WSiA (≤ 1 × 10(-3)/4 × 10(-6) mol/l), while antagonistic effect was obtained for all higher concentrations of the inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1979-01-01

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

Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

International Nuclear Information System (INIS)

Bajaj, Mamta; Moriyama, Hideaki

2007-01-01

The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V M of 1.8 Å 3 Da −1

Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

Energy Technology Data Exchange (ETDEWEB)

Bajaj, Mamta [School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States); Moriyama, Hideaki, E-mail: hmoriyama2@unl.edu [Department of Chemistry, e-Toxicology and Biotechnology, University of Nebraska-Lincoln, Hamilton Hall, Lincoln, Nebraska 68588-0304 (United States); School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States)

2007-05-01

The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V{sub M} of 1.8 Å{sup 3} Da{sup −1}.

Studies on Transcriptional Incorporation of 5'-N-Triphosphates of 5'-Amino-5'-Deoxyribonucleosides.

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Weronika Kotkowiak

Full Text Available In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleosides (5'NH NTPs. The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5'NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5'NH NTPs incorporation. Based on experimental data it is postulated that 5'NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleoside and the exact mechanism of transcriptional inhibition.

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay.

Science.gov (United States)

Calil, Felipe Antunes; Lima, Juliana Maria; de Oliveira, Arthur Henrique Cavalcante; Mariotini-Moura, Christiane; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lucia

2016-01-01

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the Tc NTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L -1 , which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100  µ mol·L -1 , which is in accordance with the data for the enzyme in solution.

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay

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Felipe Antunes Calil

2016-01-01

Full Text Available The use of IMERs (Immobilized Enzyme Reactors as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1 from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors. A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave KM of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.

Role of the ectonucleotidase NTPDase2 in taste bud function.

Science.gov (United States)

Vandenbeuch, Aurelie; Anderson, Catherine B; Parnes, Jason; Enjyoji, Keiichi; Robson, Simon C; Finger, Thomas E; Kinnamon, Sue C

2013-09-03

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.

Microcontroller-assisted compensation of adenosine triphosphate levels: instrument and method development.

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Hu, Jie-Bi; Chen, Ting-Ru; Chen, Yu-Chie; Urban, Pawel L

2015-01-30

In order to ascertain optimum conditions for biocatalytic processes carried out in vitro, we have designed a bio-opto-electronic system which ensures real-time compensation for depletion of adenosine triphosphate (ATP) in reactions involving transfer of phosphate groups. The system covers ATP concentration range of 2-48 μM. The report demonstrates feasibility of the device operation using apyrase as the ATP-depleting enzyme.

Intercalation of gaseous thiols and sulfides into Ag+ ion-exchanged aluminum dihydrogen triphosphate.

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Hayashi, Aki; Saimen, Hiroki; Watanabe, Nobuaki; Kimura, Hitomi; Kobayashi, Ayumi; Nakayama, Hirokazu; Tsuhako, Mitsutomo

2005-08-02

Ag(+) ion-exchanged layered aluminum dihydrogen triphosphate (AlP) with the interlayer distance of 0.85 nm was synthesized by the ion-exchange of proton in triphosphate with Ag(+) ion. The amount of exchanged Ag(+) ion depended on the concentration of AgNO(3) aqueous solution. Ag(+) ion-exchanged AlP adsorbed gaseous thiols and sulfides into the interlayer region. The adsorption amounts of thiols were more than those of sulfides, thiols with one mercapto group > thiol with two mercapto groups > sulfides, and depended on the amount of exchanged Ag(+) ion in the interlayer region. The thiols with one mercapto group were intercalated to expand the interlayer distance of Ag(+) ion-exchanged AlP, whereas there was no expansion in the adsorption of sulfide. In the case of thiol with two mercapto groups, there was observed contraction of the interlayer distance through the bridging with Ag(+) ions of the upper and lower sides of the interlayer region.

Hybrid integrated biological-solid-state system powered with adenosine triphosphate

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Roseman, Jared M.; Lin, Jianxun; Ramakrishnan, Siddharth; Rosenstein, Jacob K.; Shepard, Kenneth L.

2015-12-01

There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na+/K+ adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 106 mm-2) are able to sustain a short-circuit current of 32.6 pA mm-2 and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm-2 from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

Synthesis of adenosine triphosphate tritiated in position 2 and 8

International Nuclear Information System (INIS)

Cossery, Jean-Michel

1986-01-01

Adenosine triphosphate or ATP is an important molecule present at the cellular level in many fundamental biochemical mechanism, and the study of its metabolism is therefore of particular interest. In this thesis for pharmacy graduation, the author first describes the different steps of synthesis and purification leading to chloride-2-ATP, a precursor of the final tritiated molecule. Then, the author explains the tritiation of this molecule to obtain an ATP tritiated in position 2 and in position 8 [fr

Caffeic acid treatment alters the extracellular adenine nucleotide hydrolysis in platelets and lymphocytes of adult rats.

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Anwar, Javed; Spanevello, Roselia Maria; Pimentel, Victor Camera; Gutierres, Jessié; Thomé, Gustavo; Cardoso, Andreia; Zanini, Daniela; Martins, Caroline; Palma, Heloisa Einloft; Bagatini, Margarete Dulce; Baldissarelli, Jucimara; Schmatz, Roberta; Leal, Cláudio Alberto Martins; da Costa, Pauline; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

2013-06-01

This study evaluated the effects of caffeic acid on ectonucleotidase activities such as NTPDase (nucleoside triphosphate diphosphohydrolase), Ecto-NPP (nucleotide pyrophosphatase/phosphodiesterase), 5'-nucleotidase and adenosine deaminase (ADA) in platelets and lymphocytes of rats, as well as in the profile of platelet aggregation. Animals were divided into five groups: I (control); II (oil); III (caffeic acid 10 mg/kg); IV (caffeic acid 50 mg/kg); and V (caffeic acid 100 mg/kg). Animals were treated with caffeic acid diluted in oil for 30 days. In platelets, caffeic acid decreased the ATP hydrolysis and increased ADP hydrolysis in groups III, IV and V when compared to control (P<0.05). The 5'-nucleotidase activity was decreased, while E-NPP and ADA activities were increased in platelets of rats of groups III, IV and V (P<0.05). Caffeic acid reduced significantly the platelet aggregation in the animals of groups III, IV and V in relation to group I (P<0.05). In lymphocytes, the NTPDase and ADA activities were increased in all groups treated with caffeic acid when compared to control (P<0.05). These findings demonstrated that the enzymes were altered in tissues by caffeic acid and this compound decreased the platelet aggregation suggesting that caffeic acid should be considered a potentially therapeutic agent in disorders related to the purinergic system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

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Bettendorff, Lucien; Wins, Pierre

2009-06-01

Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

Fludarabine-mediated circumvention of cytarabine resistance is associated with fludarabine triphosphate accumulation in cytarabine-resistant leukemic cells.

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Yamamoto, Shuji; Yamauchi, Takahiro; Kawai, Yasukazu; Takemura, Haruyuki; Kishi, Shinji; Yoshida, Akira; Urasaki, Yoshimasa; Iwasaki, Hiromichi; Ueda, Takanori

2007-02-01

The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5'-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5'-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

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Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

2016-10-12

Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

Synthesis and study of the triphosphate salt LiSr2P3O10·8H2O

International Nuclear Information System (INIS)

Sotnikova-Yuzhik, V.A.; Peslyak, G.V.

1995-01-01

Lithium triphosphate interaction with strontium nitrate in aqueous solution at 0.3 mole% concentration and 20 deg C is studied. Formation of crystal hydrate LiSr 2 P 3 O 10 ·8H 2 O and amorphous phase of variable composition Li 2,5-0,5x P 3 O 10 ·6H 2 O (0.20≤x≤0.55) is determined. Data on the stability of binary lithium-strontium triphosphate at storage, sequence of chemical and phase transitions under heating are obtained. 5 refs., 4 figs., 3 tabs

Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli.

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Pezo, Valérie; Hassan, Camille; Louis, Dominique; Sargueil, Bruno; Herdewijn, Piet; Marlière, Philippe

2018-05-18

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

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Wenjing Qi

2016-10-01

Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

Hybrid integrated biological-solid-state system powered with adenosine triphosphate.

Science.gov (United States)

Roseman, Jared M; Lin, Jianxun; Ramakrishnan, Siddharth; Rosenstein, Jacob K; Shepard, Kenneth L

2015-12-07

There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na(+)/K(+) adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 10(6) mm(-2)) are able to sustain a short-circuit current of 32.6 pA mm(-2) and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm(-2) from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

An Escherichia coli strain deficient for both exonuclease 5 and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

International Nuclear Information System (INIS)

Estevenon, A.M.; Kooistra, J.; Sicard, N.

1995-01-01

An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity

Effects of chronic digitalization on cardiac and renal Na+ + K+-dependent adenosine triphosphate activity and circulating catecholamines in the dog.

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Nechay, B R; Jackson, R E; Ziegler, M G; Neldon, S L; Thompson, J D

1981-09-01

To extend our understanding of the mechanism of action of digitalis drugs, we studied electrocardiograms (ECGs), renal function, plasma concentrations of catecholamines, and myocardial and renal Na+ + K+-dependent adenosine triphosphate (Na+ + K+ ATPase) activity in chronically digitalized dogs. Five healthy, male, mongrel dogs received a therapeutic regimen of digoxin (0.1 mg/kg on day 1 in three divided doses followed by 0.025 mg/kg per day) orally for 2-4 months. This resulted in plasma digoxin concentrations of 1.1 to 4.7 ng/ml as determined by radioimmunoassay. Six control dogs received daily gelatin capsules by mouth. ECGs monitored throughout the study showed no changes. Digitalized dogs had elevated plasma norepinephrine concentrations (347 vs. 137 pg/ml in controls) and no change in plasma epinephrine concentrations. Digitalized dogs had elevated glomerular filtration rates (0.74 vs. 0.94 ml/min per g of kidney) without significant changes in renal handling of electrolytes and water. All of the above studies were done without the aid of restraining drugs or infusions. The animals were killed with an overdose of pentobarbital for in vitro studies. In digitalized dogs, microsomal Na+ + K+ ATPase-specific activity was 26 to 33% lower in the renal cortex, medulla, and papilla, and 46% lower in the cardiac left ventricle than in control dogs. Digitalization did not alter the osmolalities of renal tissues. We conclude that chronic reduction Na+ + K+ ATPase activity by one-third dose does not cause abnormalities in renal handling of electrolytes and water, and inhibition of Na+ + K+ ATPase in the left ventricular muscle by one-half is associated with no obvious ECG changes in the dog. Further, elevated plasma norepinephrine concentrations may contribute to both the therapeutic and the toxic effects of digitalis.

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

International Nuclear Information System (INIS)

Ide, H.; Melamede, R.J.; Wallace, S.S.

1987-01-01

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag 2 O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [ 3 H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The result suggests that Escherichia coli DNA polymerase I uses both isomers of DHdTTP as substrates and that the overall efficiency of incorporation is primarily determined by the concentration of the isomers in the nucleotide pool

Determination of adenosine disodium triphosphate using prulifloxacin-terbium(III) as a fluorescence probe by spectrofluorimetry

International Nuclear Information System (INIS)

Yu Fengshan; Li Lin; Chen Fang

2008-01-01

A new spectrofluorimetric method is developed for determination of adenosine disodium triphosphate (ATP). The interactions between prulifloxacin (PUFX)-Tb 3+ complex and adenosine disodium triphosphate has been studied by using UV-vis absorption and fluorescence spectra. Using prulifloxacin-Tb 3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the prulifloxacin-Tb 3+ complex at λ = 545 nm and the enhanced fluorescence intensity is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 4.0 x 10 -7 to 2.0 x 10 -5 mol L -1 , and the detection limit (3 σ/k) is 1.7 x 10 -8 mol L -1 . This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in real pharmaceutical samples. The mechanism of fluorescence enhancement of prulifloxacin-Tb 3+ complex by ATP was also discussed

Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

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Joanna Lecka

2014-01-01

Full Text Available Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (pathophysiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (Ki=14 μM. Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM completely inhibited the ATPase activity of NTPDase1 in situ as shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10–20% of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

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Amanda Piccoli Frasson

2012-03-01

Full Text Available Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum. The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

CaMKII Regulation of Cardiac Ryanodine Receptors and Inositol Triphosphate Receptors

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Emmanuel eCamors

2014-05-01

Full Text Available Ryanodine receptors (RyRs and inositol triphosphate receptors (InsP3Rs are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR, and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and posttranslational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

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Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

2017-11-01

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

An improved red blood cell additive solution maintains 2,3-diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage.

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Burger, Patrick; Korsten, Herbert; De Korte, Dirk; Rombout, Eva; Van Bruggen, Robin; Verhoeven, Arthur J

2010-11-01

Current additive solutions (ASs) for red blood cells (RBCs) do not maintain constant 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) levels during cold storage. We have previously shown that with a new AS called phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM), both 2,3-DPG and ATP could be maintained throughout storage for 35 days. In this study, the mechanism underlying the effect of PAGGGM on RBC storage was studied in more detail. By using double-erythrocytapheresis units (leukoreduced), a direct comparison could be made between the current AS saline-adenine-glucose-mannitol (SAGM) and the experimental solution PAGGGM. During cold storage, several in vitro characteristics were analyzed. In agreement with our previous findings with single RBCs, PAGGGM maintained 2,3-DPG and ATP levels for 35 days of cold storage. Furthermore, glucose consumption and lactate production were higher in PAGGGM units during the first 21 days of cold storage. Fructose-1,6-diphophate and dihydroxyacetone phosphate levels were also increased during the first 21 days of storage in PAGGGM units. These results indicate that it is likely that phosphofructokinase (PFK) activity is enhanced in PAGGGM units relative to SAGM units. After 21 days, PFK activity also decreases in PAGGGM units, but sufficient metabolic reserve in these units prevents depletion of 2,3-DPG and ATP. © 2010 American Association of Blood Banks.

Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39

International Nuclear Information System (INIS)

Zhong, Xiaotian; Buddha, Madhavan; Guidotti, Guido; Kriz, Ron; Somers, Will; Mosyak, Lidia

2008-01-01

The ecto-enzymatic domain of rat E-NTPDase1 CD39 was expressed and purified and diffraction-quality crystals of the enzyme were obtained. CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P3 2 , with unit-cell parameters a = b = 118.1, c = 81.6 Å and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 Å data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P3 2 lattice and rigid-body refined and position-minimized with PHENIX

Glyphosate-based herbicide affects biochemical parameters in Rhamdia quelen (Quoy & Gaimard, 1824 and Leporinus obtusidens (Valenciennes, 1837

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Vania Lucia Loro

Full Text Available Rhamdia quelen (silver catfish and Leporinus obtusidens (piava were exposed to a commercial formulation Roundup(r, a glyphosate-based herbicide at concentrations of 0.2 or 0.4 mg/L for 96 h. The effects of the herbicide were analyzed on the alanine aminotransferase (ALT and aspartate aminotransferase (AST activities and glucose in plasma, glucose and protein in the mucus layer, nucleotide hydrolysis in the brain, and protein carbonyl in the liver. The parameters were chosen, owing to a lack of information concerning integrated analysis, considering oxidative damage parameters, liver damage, and effects on the mucus layer composition and triphosphate diphosphohydrolase (NTPDase activities. Plasmatic glucose levels were reduced in both species, whereas the transaminase activities (ALT and AST increased after exposure to the herbicide. Herbicide exposure increased protein and glucose levels in the mucus layer in both species. There was a reduction in both NTPDase and ecto-5'-nucleotidase activity in the brain of piava, and increased enzyme activity in silver catfish at both concentrations tested. The species showed an increase in protein carbonyl in the liver after exposure to both concentrations of the glyphosate. Our results demonstrated that exposure to Roundup(r caused liver damage, as evidenced by increased plasma transaminases and liver protein carbonyl in both of the fish species studied. The mucus composition changed and hypoglycemia was detected after Roundup(r exposure in both species. Brain nucleotide hydrolysis showed a different response for each fish species studied. These parameters indicated some important and potential indicators of glyphosate contamination in aquatic ecosystems.

Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts

International Nuclear Information System (INIS)

Kaplan, J.H.; Forbush, B. III; Hoffman, J.F.

1978-01-01

2-Nitrobenzyl phosphate and 1-(2-nitro)phenylethyl phosphate have been synthesized and demonstrated to be suitable as photolabile sources of inorganic phosphate. The same protecting groups were attached to the terminal phosphate of adenosine 5'-triphosphate. These caged ATP compounds released adenosine 5'-triphosphate on illumination at 340 nm in aqueous solution and P 3 -1-(2-nitro)phenylethyl-ATP gave about a 70 percent yield in under 30 s. The unphotolyzed caged ATP was neither a substrate nor inhibitor of purified renal Na,K-ATPase (EC 3.61.3). Following photolysis in the presence of the enzyme, the liberated ATP was hydrolyzed but at an inhibited rate. The photo-dependent inhibition could be eliminated by prior addition of glutathione or bisulfite to the irradiated solution. Caged ATP was incorporated into resealed human erythrocyte ghosts prepared from red blood cells depleted of internal energy stores. While the NA : K pump was unable to use incorporated caged ATP as a substrate, the ATP liberated by photolysis activated the pump as evidenced by measurements of K-dependent, ouabain-sensitive Na efflux. Thus the caged ATP can be used as a stable source of ATP unmetabolizable by intracellular ATPases until the ATP is released following photolytic irradiation

Effects of adenosine triphosphate concentration on motor force regulation during skeletal muscle contraction

Science.gov (United States)

Wei, J.; Dong, C.; Chen, B.

2017-04-01

We employ a mechanical model of sarcomere to quantitatively investigate how adenosine triphosphate (ATP) concentration affects motor force regulation during skeletal muscle contraction. Our simulation indicates that there can be negative cross-bridges resisting contraction within the sarcomere and higher ATP concentration would decrease the resistance force from negative cross-bridges by promoting their timely detachment. It is revealed that the motor force is well regulated only when ATP concentration is above a certain level. These predictions may provide insights into the role of ATP in regulating coordination among multiple motors.

Molecular structure of tetraaqua adenosine 5'-triphosphate aluminium(III) complex: A study involving Raman spectroscopy, theoretical DFT and potentiometry

Science.gov (United States)

Tenório, Thaís; Silva, Andréa M.; Ramos, Joanna Maria; Buarque, Camilla D.; Felcman, Judith

2013-03-01

The Alzheimer's disease is one of the most common neurodegenerative diseases that affect elderly population, due to the formation of β-amyloid protein aggregate and several symptoms, especially progressive cognitive decline. The result is a decrease in capture of glucose by cells leading to obliteration, meddling in the Krebs cycle, the principal biochemical route to the energy production leading to a decline in the levels of adenosine 5'-triphosphate. Aluminium(III) is connected to Alzheimer's and its ion provides raise fluidity of the plasma membrane, decrease cell viability and aggregation of amyloid plaques. Studies reveal that AlATP complex promotes the formation of reactive fibrils of β-amyloid protein and independent amyloidogenic peptides, suggesting the action of the complex as a chaperone in the role pathogenic process. In this research, one of complexes formed by Al(III) and adenosine 5'-triphosphate in aqueous solution is analyzed by potentiometry, Raman spectroscopy and ab initio calculations. The value of the log KAlATP found was 9.21 ± 0.01 and adenosine 5'-triphosphate should act as a bidentate ligand in the complex. Raman spectroscopy and potentiometry indicate that donor atoms are the oxygen of the phosphate β and the oxygen of the phosphate γ, the terminal phosphates. Computational calculations using Density Functional Theory, with hybrid functions B3LYP and 6-311++G(d,p) basis set regarding water solvent effects, have confirmed the results. Frontier molecular orbitals, electrostatic potential contour surface, electrostatic potential mapped and Mulliken charges of the title molecule were also investigated.

UP-REGULATION OF ANTITHROMBOTIC ECTONUCLEOTIDASES BY ASPIRIN IN HUMAN ENDOTHELIAL-CELLS IN-VITRO

NARCIS (Netherlands)

CHEUNG, PK; VISSER, J; BAKKER, WW

1994-01-01

Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP

Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors

DEFF Research Database (Denmark)

Johansen, Torben

1979-01-01

The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely re...

Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

Science.gov (United States)

Frasson, Amanda Piccoli; De Carli, Geraldo Attilio; Bonan, Carla Denise; Tasca, Tiana

2012-03-01

Trichomonas vaginalis is a parasite from the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. The neutrophil infiltration has been considered to be primarily responsible for cytological changes observed at infection site, and the chemoattractants can play an important role in this leukocytic recruitment. Nitric oxide (NO) is one of the most widespread mediator compounds, and it is implicated in modulation of immunological mechanisms. Extracellular nucleotides and nucleosides are signaling molecules involved in several processes, including immune responses and control of leukocyte trafficking. Ectonucleoside triphosphate diphosphohydrolase members, ecto-5'-nucleotidase, and adenosine deaminase (ectoADA) have been characterized in T. vaginalis. Herein, we investigated the effects of purinergic system on NO production by neutrophils stimulated with T. vaginalis. The trophozoites were able to induce a high NO synthesis by neutrophils through iNOS pathway. The extracellular nucleotides ATP, ADP, and ATPγS (a non-hydrolyzable ATP analog) showed no significant change in NO secretion. In contrast, adenosine and its degradation product, inosine, promoted a low production of the compound. The immunosuppressive effect of adenosine upon NO release by neutrophils occurred due to adenosine A(2A) receptor activation. The ecto-5'-nucleotidase activity displayed by T. vaginalis was shown to be important in adenosine generation, indicating the efficiency of purinergic cascade. Our data suggest the influence of purinergic signaling, specifically adenosinergic system, on NO production by neutrophils in T. vaginalis infection, contributing to the immunological aspects of disease.

Biphasic Elimination of Tenofovir Diphosphate and Nonlinear Pharmacokinetics of Zidovudine Triphosphate in a Microdosing Study

Science.gov (United States)

Chen, Jianmeng; Flexner, Charles; Liberman, Rosa G.; Skipper, Paul L.; Louissaint, Nicolette; Tannenbaum, Steven R.; Hendrix, Craig; Fuchs, Edward

2012-01-01

Objective Phase 0 studies can provide initial pharmacokinetics (PK) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of two antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. Study Design We administered a microdose (100 μg) of 14C-labeled drug (ZDV or tenofovir disoproxil fumarate (TDF)) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in PBMCs and CD4+ cells were measured by AMS. Results The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 μg to 300 mg), while the intracellular TFV-DP PK were linear over the same dose range. ZDV-TP concentrations were lower in CD4+ cells versus total peripheral blood mononuclear cells (PBMCs), while TFV-DP concentrations were not different in CD4+ cells and PBMCs. Conclusion Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. AMS shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs. PMID:23187888

Adsorption characteristics of 14C-labeled alanine, aspartic acid and adenosine triphosphate by metal-chelating resins

International Nuclear Information System (INIS)

Ishiyama, Toshio; Matsunami, Tadao; Shibata, Setsuko; Honda, Yoshihide.

1987-01-01

(1) Adsorption properties of 14 C-alanine, 14 C-ATP (adenosine triphosphate) and 14 C-aspartic acid on the metal-chelating resins were determined and found that the Cu(II)-Chelex 100 and Fe(III)-Unicellex UR10, Fe(III)-Chelex 100 chelating resins were highly effective for the adsorption of 14 C-alanine and 14 C-ATP, respectively. (2) Desorption rate of 14 C-ATP from the Fe(III)-Unicellex UR10 and Fe(III)-Chelex 100 resins was somewhat higher than the case of 14 C-alanine, probably because the coordination bonds of Cu-alanine might be stronger than those of Fe-ATP. Thus, 14 C-labeled organic compounds such as 14 C-alanine and 14 C-ATP of a low activity concentration (3.7 mBq/ml) (1 x 10 -7 μCi/ml) in aqueous solution may be measured with liquid scintillation counter after pre-concentration by use of the Fe(III)- and Cu(II)-chelating resin columns. (author)

Increased deoxythymidine triphosphate levels is a feature of relative cognitive decline

DEFF Research Database (Denmark)

Madsen, Claus Desler; Frederiksen, Jane H; Olsen, Maria Nathalie Angleys

2015-01-01

Mitochondrial bioenergetics, mitochondrial reactive oxygen species (ROS) and cellular levels of nucleotides have been hypothesized as early indicators of Alzheimer's disease (AD). Utilizing relative decline of cognitive ability as a predictor of AD risk, we evaluated the correlation between change...... of cognitive ability and mitochondrial bioenergetics, ROS and cellular levels of deoxyribonucleotides. Change of cognitive abilities, scored at ages of approximately 20 and 57 was determined for a cohort of 1985 male participants. Mitochondrial bioenergetics, mitochondrial ROS and whole-cell levels...... of deoxyribonucleotide triphosphates were measured in peripheral blood mononuclear cells (PBMCs) from a total of 103 selected participants displaying the most pronounced relative cognitive decline and relative cognitive improvement. We show that relative cognitive decline is associated with higher PBMC content...

Mitochondrially-Encoded Adenosine Triphosphate Synthase 6 Gene Haplotype Variation among World Population during 2003-2013

OpenAIRE

Steven Steven; Yoni F Syukriani; Julius B Dewanto

2016-01-01

Background: Adaptation and natural selection serve as an important part of evolution. Adaptation in molecular level can lead to genetic drift which causes mutation of genetic material; one of which is polymorphism of mitochondrial DNA (mtDNA). The aim of this study is to verify the polymorphism of mitochondrially-encoded Adenosine Triphosphate synthase6gene (MT-ATP6) as one of mtDNA building blocks among tropic, sub-tropic, and polar areas. Methods: This descriptive quantitative research used...

Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

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Jerome Mauris

2009-09-01

Full Text Available Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

Energy Technology Data Exchange (ETDEWEB)

Hill, Shannon E.; Nguyen, Elaine; Ukachukwu, Chiamaka U.; Freeman, Dana M.; Quirk, Stephen; Lieberman, Raquel L.; Boggon, Titus J.

2017-07-25

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

Directory of Open Access Journals (Sweden)

Shannon E Hill

Full Text Available Dihydroneopterin triphosphate pyrophosphatase (DHNTPase, a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

Science.gov (United States)

Mitamura, Toshiaki; Yamamura, Yoshimi; Kurosaki, Fumiya

2011-01-01

Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

Czech Academy of Sciences Publication Activity Database

Balintová, Jana; Simonova, Anna; Bialek-Pietras, M.; Olejniczak, A.; Lesnikowski, Z. J.; Hocek, Michal

2017-01-01

Roč. 27, č. 21 (2017), s. 4786-4788 ISSN 0960-894X R&D Projects: GA ČR GBP206/12/G151 Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : nucleotides * nucleoside triphosphates * carboranes * DNA polymerase * oligonucleotides Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.454, year: 2016

Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: adenosylcobalamin destruction and formation of a nucleotide-based radical.

Science.gov (United States)

Lohman, Gregory J S; Gerfen, Gary J; Stubbe, Joanne

2010-02-23

Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP) in cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [1'-(2)H]F(2)CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence.

NARCIS (Netherlands)

Pellegrini, P.; Sauerwein, R.W.; Finlayson, T.; McLeod, J.; Covell, D.A.; Maier, T.; Machida, C.A.

2009-01-01

INTRODUCTION: Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate

Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA

International Nuclear Information System (INIS)

Evans, R.K.; Haley, B.E.

1987-01-01

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N 3 dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N 3 dUTP is the nitration of dUMP in 98% yield in 5 min at 25 0 C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH 2 dUMP). Diazotization of 5-NH 2 dUMP with HNO 2 followed by the addition NaN 3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N 3 dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N 3 dUTP through a chemical coupling of pyrophosphate to 5-N 3 dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N 3 UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [γ- 32 P]-5-N 3 dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing γ- 32 P-labeled pyrimidine nucleotides was developed. [γ- 32 P]-5-N 3 dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP. The photoactivity of 5-N 3 dUMP is stable to extremes of pH, and [γ- 32 P]-5-N 3 dUTP was an effective photolabeling reagent even in the presence of 10 mM dithiothreitol

Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

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Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

2017-06-01

In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver

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Ana V. Frontini

2011-04-01

Full Text Available The utilization of adenosine 5´-triphosphate (ATP infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.

HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway

Directory of Open Access Journals (Sweden)

Ana White

2012-02-01

The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP3levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP3 signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP3 levels during the reproductive cycle.

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

International Nuclear Information System (INIS)

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

1978-01-01

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

Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

Science.gov (United States)

Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

2015-03-03

A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

Peculiarities of different-ligand complexing of rare earths with nitrilotriacetate and adenosine-5'-triphosphate according to the mathematical simulation data

International Nuclear Information System (INIS)

Svetlova, I.E.; Dobrynina, N.A.; Smirnova, N.S.; Martynenko, L.I.; Evseev, A.M.

1988-01-01

By the method of pH-metric titration using mathematical simulation different-ligand complexing of rare earths with nitrilotriacetate and adenosine-5'-triphosphate is studied. It is shown that the ligands interact with the formation of protonated associates. The composition of different complexes is determined, their stability constants are calculated, their existence regions are found

Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

Science.gov (United States)

Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

2006-09-01

A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

Free and nanoencapsulated vitamin D3 : effects on E-NTPDase and E-ADA activities in an animal model with induced arthritis.

Science.gov (United States)

da Silveira, Karine Lanes; da Silveira, Leonardo Lanes; Thorstenberg, Maria Luiza Prates; Cabral, Fernanda Licker; Castilhos, Livia Gelain; Rezer, João Felipe Peres; de Andrade, Diego Fontana; Beck, Ruy Carlos Ruver; Einloft Palma, Heloísa; de Andrade, Cinthia Melazzo; Pereira, Renata da Silva; Martins, Nara Maria Beck; Bertonchel Dos Santos, Claudia de Mello; Leal, Daniela Bitencourt Rosa

2016-06-01

The effect of vitamin D3 in oral solution (VD3 ) and vitamin D3 -loaded nanocapsules (NC-VD3 ) was analysed in animals with complete Freund's adjuvant (CFA) induced arthritis (AR). For this purpose, we evaluated scores for arthritis, thermal hyperalgesia and paw oedema, as well as histological analyses and measurements of the activity of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) enzymes in rat lymphocytes. Haematological and biochemical parameters were also determined. The doses administered were 120 UI/day of VD3 and 15.84 UI/day of NC-VD3 . Fifteen days after the induction of AR, the groups were treated for 15 days with vitamin D3 . The results demonstrated that VD3 was able to reduce arthritis scores, thermal hyperalgesia and paw oedema in rats with CFA-induced arthritis. However, treatment with NC-VD3 did not reduce arthritis scores. The histological analyses showed that both formulations were able to reduce the inflammatory changes induced by CFA. The activity of E-NTPDase in rat lymphocytes was higher in the AR compared with the control group, while the activity of E-ADA was lower. This effect was reversed after the 15-day treatment. Data from this study indicates that both forms of vitamin D3 seem to contribute to decreasing the inflammatory process induced by CFA, possibly altering the activities of ectoenzymes. Copyright © 2016 John Wiley & Sons, Ltd. The effects promoted by both formulations of vitamin D3 , either in oral solution or nanoencapsulated form, strongly suggests the softening of the inflammatory process induced by complete Freund's adjuvant (CFA), possibly altering the E-NTPDase and E-ADA activities. However, it is known that vitamin D has a beneficial effect on the modulation of the immune system components responsible for the inflammatory process. Moreover, the establishment of responses to treatment with vitamin D3 may provide an alternative for inhibiting the proinflammatory

Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

International Nuclear Information System (INIS)

Mahmood, R.

1985-01-01

The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz 2 ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz 2 ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF 1 ) with a binding constant of 3.2 x 10 5 M -1 . Bz 2 ATP is also a substrate for the ATPase activity of SF 1 in the presence of different cations. The irradiation of SF 1 with [ 3 H]Bz 2 ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF 1 after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling [ 3 H]Bz 2 ATP is trapped on SF 1 by cross-linking the two reactive thiols, SH 1 and SH 2 , by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified [ 14 C]Bz 2 ATP-SF 1 , after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography

Adenosine Triphosphate (ATP Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway

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Zhengchang Liu

2013-03-01

Full Text Available Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.

Creatine kinase activity is associated with blood pressure

NARCIS (Netherlands)

Brewster, Lizzy M.; Mairuhu, Gideon; Bindraban, Navin R.; Koopmans, Richard P.; Clark, Joseph F.; van Montfrans, Gert A.

2006-01-01

BACKGROUND: We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular

Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica.

Science.gov (United States)

Nozaki, T; Arase, T; Shigeta, Y; Asai, T; Leustek, T; Takeuchi, T

1998-12-08

A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms. The deduced amino acid sequence of E. histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38. The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp. and Bacillus subtilis (48 and 44%, respectively). Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E. histolytica AS. The upstream region of the AS gene contained three conserved elements reported for other E. histolytica genes. A recombinant E. histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions. Expression of the E. histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains. Genomic hybridization revealed that the AS gene exists as a single copy gene. In the literature, this is the first description of an AS gene in Protozoa.

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

International Nuclear Information System (INIS)

Dhar, A.; Paul, A.K.; Shukla, S.D.

1990-01-01

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

Enzymatic primer-extension with glycerol-nucleoside triphosphates on DNA templates.

Directory of Open Access Journals (Sweden)

Jesse J Chen

Full Text Available BACKGROUND: Glycerol nucleic acid (GNA has an acyclic phosphoglycerol backbone repeat-unit, but forms stable duplexes based on Watson-Crick base-pairing. Because of its structural simplicity, GNA is of particular interest with respect to the possibility of evolving functional polymers by in vitro selection. Template-dependent GNA synthesis is essential to any GNA-based selection system. PRINCIPAL FINDINGS: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitative primer-extension by the incorporation of two glyceronucleotides, with much less efficient extension up to five glyceronucleotides. Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines. In an attempt to improve pyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substituted pyrimidine nucleobases. This led to more efficient incorporation of gC, but not gT. CONCLUSIONS: We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.

Enhancement of DNA polymerase activity in potato tuber slices

International Nuclear Information System (INIS)

Watanabe, Akira; Imaseki, Hidemasa

1977-01-01

DNA polymerase was extracted from potato (Soleum tuberosum L.) tuber discs and the temporal correlation of its activity change to DNA synthesis in vivo was examined during aging of the discs. Most of the DNA polymerase was recovered as a bound form in the 18,000 x g precipitate. Reaction with the bound-form enzyme was dependent on the presence of four deoxynucleoside triphosphates, Mg 2+ , and a template. ''Activated'' DNA and heat-denatured DNA, but not native DNA, were utilized as templates. The polymerase activity was sensitive to SH reagents. Fresh discs, which do not synthesize DNA in vivo, contained a significant amount of DNA polymerase and its activity increased linearly with time until 48 hr after slicing and became four times that of fresh discs after 72 hr, whereas the activity of DNA synthesis in vivo increased with time and decreased after reaching a maximum at 30 hr. Cycloheximide inhibited the enhancement of polymerase activity. DNA polymerase from aged and fresh discs had identical requirements for deoxynucleotides and a template in their reactions, sensitivity to SH reagent, and affinity to thymidine triphosphate. (auth.)

A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

Science.gov (United States)

Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

2015-01-01

A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

SIMULTANEOUS ANALYSIS OF AZIDOTHYMIDINE AND ITS MONOPHOSPHATE, DIPHOSPHATE AND TRIPHOSPHATE DERIVATIVES IN BIOLOGICAL-FLUIDS, TISSUE AND CULTURED-CELLS BY A RAPID HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD

NARCIS (Netherlands)

MOLEMA, G; JANSEN, RW; Visser, Jan; MEIJER, DKF

1992-01-01

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro

Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

Science.gov (United States)

Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

2016-05-01

Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

Synthesis of Base-Modified 2 '-Deoxyribonucleoside Triphosphates and Their Use in Enzymatic Synthesis of Modified DNA for Applications in Bioanalysis and Chemical Biology

Czech Academy of Sciences Publication Activity Database

Hocek, Michal

2014-01-01

Roč. 79, č. 21 (2014), s. 9914-9921 ISSN 0022-3263 R&D Projects: GA ČR GBP206/12/G151; GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : cross - coupling reactions * modified nucleoside triphosphates * nucleic acids Subject RIV: CC - Organic Chemistry Impact factor: 4.721, year: 2014

Nitrogen and phosphorus co-doped graphene quantum dots: synthesis from adenosine triphosphate, optical properties, and cellular imaging.

Science.gov (United States)

Ananthanarayanan, Arundithi; Wang, Yue; Routh, Parimal; Sk, Mahasin Alam; Than, Aung; Lin, Ming; Zhang, Jie; Chen, Jie; Sun, Handong; Chen, Peng

2015-05-07

Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells.

On the structure of thorium and americium adenosine triphosphate complexes

International Nuclear Information System (INIS)

Mostapha, Sarah; Berton, Laurence; Boubals, Nathalie; Zorz, Nicole; Charbonnel, Marie-Christine; Fontaine-Vive, Fabien; Den Auwer, Christophe; Solari, Pier Lorenzo

2014-01-01

The actinides are chemical poisons and radiological hazards. One challenge to better appraise their toxicity and develop countermeasures in case of exposure of living organisms is to better assess pathways of contamination. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, nucleotides and in particular adenosine triphosphate nucleotide (ATP) may be considered critical target building blocks for actinides. Combinations of spectroscopic techniques (Fourier transformed Infra Red [FTIR], Electro-spray Ionization Mass Spectrometry [ESI-MS], and Extended X-ray Absorption Fine Structure [EXAFS]) with quantum chemical calculations have been implemented in order to assess the actinides coordination arrangement with ATP. We describe and compare herein the interaction of ATP with thorium and americium; thorium(IV) as a representative of actinide(IV) like plutonium(IV) and americium(III) as a representative of all heavier actinides. In the case of thorium, an insoluble complex is readily formed. In the case of americium, a behavior identical to that described previously for lutetium has been observed with insoluble and soluble complexes. The comparative study of ATP complexation with Th(IV) and Am(III) shows their ability to form insoluble complexes for which a structural model has been proposed by analogy with previously described Lu(III) complexes. (authors)

On the structure of thorium and americium adenosine triphosphate complexes.

Science.gov (United States)

Mostapha, Sarah; Fontaine-Vive, Fabien; Berthon, Laurence; Boubals, Nathalie; Zorz, Nicole; Solari, Pier Lorenzo; Charbonnel, Marie Christine; Den Auwer, Christophe

2014-11-01

The actinides are chemical poisons and radiological hazards. One challenge to better appraise their toxicity and develop countermeasures in case of exposure of living organisms is to better assess pathways of contamination. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, nucleotides and in particular adenosine triphosphate nucleotide (ATP) may be considered critical target building blocks for actinides. Combinations of spectroscopic techniques (Fourier transformed Infra Red [FTIR], Electrospray Ionization Mass Spectrometry [ESI-MS], and Extended X-ray Absorption Fine Structure [EXAFS]) with quantum chemical calculations have been implemented in order to assess the actinides coordination arrangement with ATP. We describe and compare herein the interaction of ATP with thorium and americium; thorium(IV) as a representative of actinide(IV) like plutonium(IV) and americium(III) as a representative of all heavier actinides. In the case of thorium, an insoluble complex is readily formed. In the case of americium, a behavior identical to that described previously for lutetium has been observed with insoluble and soluble complexes. The comparative study of ATP complexation with Th(IV) and Am(III) shows their ability to form insoluble complexes for which a structural model has been proposed by analogy with previously described Lu(III) complexes.

The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

Energy Technology Data Exchange (ETDEWEB)

Sekiya, M.; Frohlich, E.D.; Cole, F.E. (Alton Ochsner Medical Foundation, New Orleans, LA (USA))

1991-01-01

In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

OpenAIRE

Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

1995-01-01

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

Science.gov (United States)

Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

2016-01-01

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

Mesenchymal stem cells from different murine tissues have differential capacity to metabolize extracellular nucleotides.

Science.gov (United States)

Iser, Isabele C; Bracco, Paula A; Gonçalves, Carlos E I; Zanin, Rafael F; Nardi, Nance B; Lenz, Guido; Battastini, Ana Maria O; Wink, Márcia R

2014-10-01

Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically. © 2014 Wiley Periodicals, Inc.

The effect of experimental gastric dilatation-volvulus on adenosine triphosphate content and conductance of the canine gastric and jejunal mucosa

OpenAIRE

Peycke, Laura E.; Hosgood, Giselle; Davidson, Jacqueline R.; Tetens, Joanne; Taylor, H. Wayne

2005-01-01

The objective of this study was to determine if experimental gastric dilatation volvulus (GDV) would decrease adenosine triphosphate (ATP) concentration and increase membrane conductance of the canine gastric and jejunal mucosa. Male dogs (n = 15) weighing between 20 and 30 kg were used. Dogs were randomly assigned to 1 of 3 equal groups: Group 1 was control, group 2 was GDV, and group 3 was ischemia. All dogs were anesthetized for 210 min. Group 1 had no manipulation. Group 2 had GDV experim...

A novel conductometric biosensor based on hexokinase for determination of adenosine triphosphate.

Science.gov (United States)

Kucherenko, I S; Kucherenko, D Yu; Soldatkin, O O; Lagarde, F; Dzyadevych, S V; Soldatkin, A P

2016-04-01

The paper presents a simple and inexpensive reusable biosensor for determination of the concentration of adenosine-5'-triphosphate (ATP) in aqueous samples. The biosensor is based on a conductometric transducer which contains two pairs of gold interdigitated electrodes. An enzyme hexokinase was immobilized onto one pair of electrodes, and bovine serum albumin-onto another pair (thus, a differential mode of measurement was used). Conditions of hexokinase immobilization on the transducer by cross-linking via glutaraldehyde were optimized. Influence of experimental conditions (concentration of magnesium ions, ionic strength and concentration of the working buffer) on the biosensor work was studied. The reproducibility of biosensor responses and operational stability of the biosensor were checked during one week. Dry storage at -18 °C was shown to be the best conditions to store the biosensor. The biosensor was successfully applied for measurements of ATP concentration in pharmaceutical samples. The proposed biosensor may be used in future for determination of ATP and/or glucose in water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

OpenAIRE

Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

2016-01-01

Zr(IV) can form phosphate and Zr(IV) (?PO3 2??Zr4+?) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). Aft...

Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

Science.gov (United States)

Hocek, Michal

2014-11-07

The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

Simple, Fast and Selective Detection of Adenosine Triphosphate at Physiological pH Using Unmodified Gold Nanoparticles as Colorimetric Probes and Metal Ions as Cross-Linkers

Directory of Open Access Journals (Sweden)

Huan Pang

2012-11-01

Full Text Available We report a simple, fast and selective colorimetric assay of adenosine triphosphate (ATP using unmodified gold nanoparticles (AuNPs as probes and metal ions as cross-linkers. ATP can be assembled onto the surface of AuNPs through interaction between the electron-rich nitrogen atoms and the electron-deficient surface of AuNPs. Accordingly, Cu2+ ions induce a change in the color and UV/Vis absorbance of AuNPs by coordinating to the triphosphate groups and a ring nitrogen of ATP. A detection limit of 50 nM was achieved, which is comparable to or lower than that achievable by the currently used electrochemical, spectroscopic or chromatographic methods. The theoretical simplicity and high selectivity reported herein demonstrated that AuNPs-based colorimetric assay could be applied in a wide variety of fields by rationally designing the surface chemistry of AuNPs. In addition, our results indicate that ATP-modified AuNPs are less stable in Cu2+, Cd2+ or Zn2+-containing solutions due to the formation of the corresponding dimeric metal-ATP complexes.

Nonequilibrium structure and dynamics in a microscopic model of thin-film active gels

NARCIS (Netherlands)

Head, D.A.; Briels, Willem J.; Gompper, G.

2014-01-01

In the presence of adenosine triphosphate, molecular motors generate active force dipoles that drive suspensions of protein filaments far from thermodynamic equilibrium, leading to exotic dynamics and pattern formation. Microscopic modeling can help to quantify the relationship between individual

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy.

Science.gov (United States)

Cheng, Yuanhua; Hogarth, Kaley A; O'Sullivan, M Lynne; Regnier, Michael; Pyle, W Glen

2016-01-01

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM. Copyright © 2016 the American Physiological Society.

Simple detection of hepatitis C virus using {sup 125}I-2'-deoxyuridine triphosphate and gamma counter

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Lee, Soo Jin; Ahn, S. H.; Chung, W. S.; Woo, K. S.; Lim, S. J.; Choi, C. W.; Lim, S. M. [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

2000-07-01

Hepatitis C Virus (HCV) is the major cause of post transfusion and sporadic non A, non B hepatitis. Current infection of HCV can be detected by PCR method. Using PCR, it has been possible to detect HCV viremia prior to immunological sero-conversion and to detect fluctuation of viremia in antibody-positive chronic HCV patients undergoing therapy with interferon. In this study, we established the simple method to detect HCV DNA by incorporation of {sup 125}I-deoxyuridine triphosphate(dUTP) into DNA during the PCR, and counted the radioactivity of PCR product by gamma counter. {sup 125}I-2'-deoxyuridine 5'-triphosphate was prepared, and incorporated into DNA during PCR. dUTP was radiolabeled by the iododemercuration of 5-mercuri intermediate. Iododemercuration labeling was completed with 98% yield and the obtained product was incorporated into DNA without further purification. After incorporation, covalently bonded radioiodine substituent was remained stable during PCR procedure HCV positive standard and positive patient sera in immunological assay were centrifuged. HCV RNA is isolated from by GTC(Guanidine Thiocyanate) and phenol/chloroform extraction method and synthesized complementary DNA by using reverse transcriptase. The '1{sup 25}I-dUTP was incorporated into HCV C DNA during PCR. PCR product purified by fiber matrix column and counted by gamma counter. PCR products were electrophoresized, and autoradiography image obtained. Amplified HCV DNA by {sup 125}I-dUTP PCR obtained the band on the gel by electrophoresis and autoradiography at the same position. In patient sera, radioactivity of HCV positive sample was 8 times higher than HCV negative viremia sample. We established HCV detection method using {sup 125}I-dUTP. {sup 125}I-dUTP PCR detection of HCV is convenient and reporducible.

Adenosine triphosphate-guided pulmonary vein isolation for atrial fibrillation: the UNmasking Dormant Electrical Reconduction by Adenosine TriPhosphate (UNDER-ATP) trial.

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Kobori, Atsushi; Shizuta, Satoshi; Inoue, Koichi; Kaitani, Kazuaki; Morimoto, Takeshi; Nakazawa, Yuko; Ozawa, Tomoya; Kurotobi, Toshiya; Morishima, Itsuro; Miura, Fumiharu; Watanabe, Tetsuya; Masuda, Masaharu; Naito, Masaki; Fujimoto, Hajime; Nishida, Taku; Furukawa, Yoshio; Shirayama, Takeshi; Tanaka, Mariko; Okajima, Katsunori; Yao, Takenori; Egami, Yasuyuki; Satomi, Kazuhiro; Noda, Takashi; Miyamoto, Koji; Haruna, Tetsuya; Kawaji, Tetsuma; Yoshizawa, Takashi; Toyota, Toshiaki; Yahata, Mitsuhiko; Nakai, Kentaro; Sugiyama, Hiroaki; Higashi, Yukei; Ito, Makoto; Horie, Minoru; Kusano, Kengo F; Shimizu, Wataru; Kamakura, Shiro; Kimura, Takeshi

2015-12-07

Most of recurrent atrial tachyarrhythmias after pulmonary vein isolation (PVI) for atrial fibrillation (AF) are due to reconnection of PVs. The aim of the present study was to evaluate whether elimination of adenosine triphosphate (ATP)-induced dormant PV conduction by additional energy applications during the first ablation procedure could reduce the incidence of recurrent atrial tachyarrhythmias. We randomly assigned 2113 patients with paroxysmal, persistent, or long-lasting AF to either ATP-guided PVI (1112 patients) or conventional PVI (1001 patients). The primary endpoint was recurrent atrial tachyarrhythmias lasting for >30 s or those requiring repeat ablation, hospital admission, or usage of Vaughan Williams class I or III antiarrhythmic drugs at 1 year with the blanking period of 90 days post ablation. Among patients assigned to ATP-guided PVI, 0.4 mg/kg body weight of ATP provoked dormant PV conduction in 307 patients (27.6%). Additional radiofrequency energy applications successfully eliminated dormant conduction in 302 patients (98.4%). At 1 year, 68.7% of patients in the ATP-guided PVI group and 67.1% of patients in the conventional PVI group were free from the primary endpoint, with no significant difference (adjusted hazard ratio [HR] 0.89; 95% confidence interval [CI] 0.74-1.09; P = 0.25). The results were consistent across all the prespecified subgroups. Also, there was no significant difference in the 1-year event-free rates from repeat ablation for any atrial tachyarrhythmia between the groups (adjusted HR 0.83; 95% CI 0.65-1.08; P = 0.16). In the catheter ablation for AF, we found no significant reduction in the 1-year incidence of recurrent atrial tachyarrhythmias by ATP-guided PVI compared with conventional PVI. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate

International Nuclear Information System (INIS)

Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

2014-01-01

We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm 2  V −1  s −1 . The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM. (paper)

Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate.

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Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

2014-04-25

We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm2 V(-1) s(-1). The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM.

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

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Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

International Nuclear Information System (INIS)

Koka, P.

1984-01-01

A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

A label-free fluorescent adenosine triphosphate biosensor via overhanging aptamer-triggered enzyme protection and target recycling amplification.

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Wang, Zhaoyin; Zhao, Jian; Dai, Zhihui

2016-06-20

Herein, a label-free fluorescent adenosine triphosphate (ATP) aptasensor is fabricated with a DNA hairpin and an overhanging aptamer. In the presence of ATP, the overhanging sequences of the aptamer may form preferred substrates of exo III, and thus trigger the enzyme-assisted amplification, which results in the release of G-rich sequences. Free G-rich sequences subsequently generate an enhanced flourescent signal by binding with thioflavin T. However, if ATP is absent, the overhanging sequence can induce steric hindrance and protect the DNA hairpin against the digestion of exo III, significantly reducing the noise of this biosensor. Accordingly, the signal-to-noise ratio of the sensing system is greatly improved, which ensures the desirable analytical performance of the proposed aptasensor both in pure samples and real samples.

Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios

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Dorris David

2002-07-01

Full Text Available Abstract Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. Conclusions Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.

Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

International Nuclear Information System (INIS)

Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

1987-01-01

The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

Science.gov (United States)

Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

1975-01-01

Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

Effect molybdenum (5, 6) compounds on hydrolysis and reorganization rates of ammonium and potassium triphosphates in aqueous solutions

International Nuclear Information System (INIS)

Petrovskaya, L.I.; Prodan, E.A.; Lesnikovich, L.A.

1992-01-01

Method of thin-layer chromatography was used to investigate the kinetics of hydrolytic decomposition of triphosphate-anion in aqueous solutions of M 5 P 3 O 10 -(NH 4 ) 6 xMo 7 O 24 -H 2 O systems (M - NH 4 , pH 5.9-6.4; M - K, pH 6.5-6.8) and (NH 4 ) 5 P 3 O 10 -(NH 4 ) 2 MoOCl 5 -H 2 O system (pH 2.5 and 4.5) at 20 deg C and 0.2 mol% concnetration. It is shown that oxoions Mo 5 produce less accelerating effect on hydrolysis, as compared to Mo 6 oxoions, and are able to initiate anion reorganization with formation of tetraphosphate under certain conditions

Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

International Nuclear Information System (INIS)

Dreslor, S.L.; Frattini, M.G.

1987-01-01

Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

Science.gov (United States)

Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

2005-01-01

Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes*

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Hindman, Ryan; Gollnick, Paul

2016-01-01

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5′ to 3′ translocase on single-stranded DNA. PMID:27189950

Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187

DEFF Research Database (Denmark)

Johansen, Torben

1979-01-01

The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast...... cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p......-trifluorometroxyphenylnydrazone), which are known to block the energy-dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187-induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5...

Visual and light scattering spectrometric method for the detection of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles

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Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

2017-02-01

A highly selective method was presented for colorimetric determination of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

As to the clastogenic-, sister-chromatid exchange inducing-and cytotoxic activity of inosine triphosphate in cultures of human peripheral lymphocytes.

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Vormittag, W; Brannath, W

2001-05-09

The influence of commercial inosine triphosphate (ITP) on the chromosome aberration rate, the mitotic rate, sister-chromatid exchange (SCE) frequency, and the proportion of first (X1), second (X2) and third (X3) division metaphases was investigated in 72h cultures of human peripheral lymphocytes. The blood donors had mild inactive arthrosis and a normal health check-up. All cultures of each volunteer were set-up simultaneously. In contrast to a previous report [Arch. Biochem. Biophys. 278 (1990) 238-244], it was demonstrated in two preliminary studies (number of subjects, n=5 each) that ITP at a final concentration of 100 microM does not induce chromosomal aberrations and, furthermore, that not ITP concentrations higher than 100 microM but ITP doses higher than 3.8mM prohibit culture growth. Based on these results, cultures with a final ITP concentration of 3.6mM (max.) and 1.8mM (max./2) were compared with control cultures (number of subjects n=10; three males and seven females, mean age x=57.6 years). Whereas no increase in the chromosomal breakage rate was observed in cultures with an ITP concentration of 1.8mM and only a marginally significant one (P=0.048) for 3.6mM ITP cultures, a highly significant induction of SCEs, not only at an ITP concentration of 3.6mM (Prate from 0 to 1.8mM as well as from 1.8 to 3.6mM in the aberration studies (all P values are equal to smallest possible one for a sample size of 10, namely, 0.002), and in the SCE studies there is a significant decrease in the X3 frequency when ITP is increased (0-1.8mM: P=0.0061 and 1.8-3.6mM: Pchanges significantly only at the second dose step (0-1.8mM ITP: P=0.22 and 1.8-3.6mM ITP: P<0.0001). The results are discussed.

Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

International Nuclear Information System (INIS)

Dowling, J.N.; Saha, A.K.; Glew, R.H.

1987-01-01

Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP 3 ) was explored. When neutrophil phosphoinositides were labeled with 32 P, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP 2 ) over 2 h. Treatment of [ 3 H]inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP 2 . Following fMLP stimulation, the fractional reduction in PIP 2 and the fractional increase in IP 3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP 3 was reduced by ACP pre-treatment. The reduction in IP 3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP 2 available for hydrolysis. However, some loss of IP 3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP 2 , the prognitor of IP 3 , and by hydrolyzing IP 3 itself

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

International Nuclear Information System (INIS)

Hamel, E.; Batra, J.K.; Lin, C.M.

1986-01-01

Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

Science.gov (United States)

Omori, Takashi; Idehara, Kenji; Kojima, Hajime; Sozu, Takashi; Arima, Kazunori; Goto, Hirohiko; Hanada, Tomohiko; Ikarashi, Yoshiaki; Inoda, Taketo; Kanazawa, Yukiko; Kosaka, Tadashi; Maki, Eiji; Morimoto, Takashi; Shinoda, Shinsuke; Shinoda, Naoki; Takeyoshi, Masahiro; Tanaka, Masashi; Uratani, Mamoru; Usami, Masahito; Yamanaka, Atsushi; Yoneda, Tomofumi; Yoshimura, Isao; Yuasa, Atsuko

2008-01-01

The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

The clinical value of adenosine triphosphate stress myocardial perfusion tomography for detecting coronary artery disease

International Nuclear Information System (INIS)

Yao Zhiming; He Qing; Qu Wanying; Yu Xue; Han Lijun; Yu Zhiguo; Li Wei; Zeng Xuezhai; Zhu Ming; Zhao Hongshan

2002-01-01

Objective: To study the clinical value of adenosine triphosphate stress myocardial perfusion tomography imaging (ATP-MPI) in detection of coronary artery disease (CAD). Methods: There were 278 patients underwent ATP-MPI, 51 patients of them also underwent coronary angiography (CAG). Seventy-three patients underwent stress-rest myocardial perfusion tomography imaging with multi-stage submaximal exercise test (ST-MPI) and CAG serving as control group. Results: 1) Side effects: there were 11 different symptoms and atrioventricular conduction block (10 patients), sinoatrial conduction block (2 patients) occurred during ATP stress. Allopathy or interruption of ATP stress did not happen. 2) The sensitivity and specificity of ATP-MPI in detection of CAD were 97.1% and 82.4%, respectively, and those in detection of ≥50% narrowing coronary artery were 91.0% and 94.7%, respectively. 3) In patients without myocardial infarction, the sensitivity and specificity of ATP-MPI in detection of myocardial ischemia were comparable to those of ST-MPI. Conclusion: ATP-MPI is an accurate, safe modality and is comparable to ST-MPI in the detection of CAD

Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus

DEFF Research Database (Denmark)

Christoffersen, Stig; Kadziola, Anders; Johansson, Eva

2009-01-01

and PPi, in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme...

Restoration of uridine 5′-triphosphate-suppressed delayed rectifying K+ currents by an NO activator KMUP-1 involves RhoA/Rho kinase signaling in pulmonary artery smooth muscle cells

Directory of Open Access Journals (Sweden)

Zen-Kong Dai

2016-12-01

Full Text Available We have demonstrated that KMUP-1 (7-[2-[4-(2-chlorobenzenepiperazinyl]ethyl]-1,3-dimethylxanthine blunts monocrotaline-induced pulmonary arterial hypertension by altering Ca2+ sensitivity, K+-channel function, endothelial nitric oxide synthase activity, and RhoA/Rho kinase (ROCK expression. This study further investigated whether KMUP-1 impedes uridine 5′-triphosphate (UTP-inhibited delayed rectifying K+ (KDR current in rat pulmonary arteries involved the RhoA/ROCK signaling. Pulmonary artery smooth muscle cells (PASMCs were enzymatically dissociated from rat pulmonary arteries. KMUP-1 (30μM attenuated UTP (30μM-mediated membrane depolarization and abolished UTP-enhanced cytosolic Ca2+ concentration. Whole-cell patch-clamp electrophysiology was used to monitor KDR currents. A voltage-dependent KDR current was isolated and shown to consist of a 4-aminopyridine (5mM-sensitive component and an insensitive component. The 4-aminopyridine sensitive KDR current was suppressed by UTP (30μM. The ROCK inhibitor Y27632 (30μM abolished the ability of UTP to inhibit the KDR current. Like Y27632, KMUP-1 (30μM similarly abolished UTP-inhibited KDR currents. Superfused protein kinase A and protein kinase G inhibitors (KT5720, 300nM and KT5823, 300nM did not affect UTP-inhibited KDR currents, but the currents were restored by adding KMUP-1 (30μM to the superfusate. KMUP-1 reversal of KDR current inhibition by UTP predominantly involves the ROCK inhibition. The results indicate that the RhoA/ROCK signaling pathway plays a key role in eliciting PASMCs depolarization caused by UTP, which would result in pulmonary artery constriction. KMUP-1 blocks UTP-mediated PASMCs depolarization, suggesting that it would prevent abnormal pulmonary vasoconstriction.

Separation of DNA-dependent polymerate activities in Micrococcus radiodurans

International Nuclear Information System (INIS)

Kitayama, S.; Matsuyama, A.

1977-01-01

DNA polymerase activities in Micrococcus radiodurans were separated into two fractions after purification more than 2000 fold. They differ in pH optimum and residual activities in the absence of a full deoxyribonucleoside triphosphates complement. NAD partly inhibited one of the activities. Both activities were eluted as a single peak on gel filtration and sedimented at the same rate on glycerol gradient centrifugation. Molecular weight 140000 was calculated from Stokes radius and sedimentation constant. Deoxyribonuclease activity was detected on one of the polymerase activities which preferentially degraded double-stranded DNA. Priming activity of nicked DNA was reduced by γ-radiation. These results have been related to the possible roles in repair synthesis in vivo or DNA synthesis in permeable cells of M. radiodurans

Separation of DNA-dependent polymerase activities in Micrococcus radiodurans

Energy Technology Data Exchange (ETDEWEB)

Kitayama, S; Matsuyama, A [Institute of Physical and Chemical Research, Wako, Saitama (Japan)

1977-03-02

DNA polymerase activities in Micrococcus radiodurans were separated into two fractions after purification more than 2000 fold. They differ in pH optimum and residual activities in the absence of a full deoxyribonucleoside triphosphates complement. NAD partly inhibited one of the activities. Both activities were eluted as a single peak on gel filtration and sedimented at the same rate on glycerol gradient centrifugation. Molecular weight 140000 was calculated from Stokes radius and sedimentation constant. Deoxyribonuclease activity was detected on one of the polymerase activities which preferentially degraded double-stranded DNA. Priming activity of nicked DNA was reduced by ..gamma.. radiation. These results have been related to the possible roles in repair synthesis in vivo or DNA synthesis in permeable cells of M. radiodurans.

Influence of morphology and topography on potentiometric response of magnesium and calcium sensitive PEDOT films doped with adenosine triphosphate (ATP)

International Nuclear Information System (INIS)

Paczosa-Bator, B.; Peltonen, J.; Bobacka, J.; Lewenstam, A.

2006-01-01

Poly(3,4-ethylenedioxythiophene) (PEDOT) films doped with adenosine triphosphate (ATP) are used to study the biologically relevant competitive magnesium and calcium ion-exchange at ATP membrane sites. It is shown, by atomic force microscopy (AFM) and scanning electron microscopy (SEM), that the surface topography and morphology of the PEDOT-ATP films determines the quality of their potentiometric response. More smooth and less rough films result in better potentiometric characteristics, particularly in a faster response. The topography/morphology of the PEDOT-ATP films is influenced by conditions during electrodeposition (electrochemical method of deposition, pH, concentration of electrolytes) and post-deposition soaking (including net-time of soaking), as evidenced by X-ray photoelectron spectroscopy (XPS) and energy dispersive analysis of X-rays (EDAX)

Heterogeneity of tumor chemosensitivity in ovarian epithelial cancer revealed using the adenosine triphosphate-tumor chemosensitivity assay.

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Zhang, Jin; Li, Hongxia

2015-05-01

Ovarian cancer has a poor prognosis, primarily due to the heterogeneity in chemosensitivity among patients. In the present study, this heterogeneity was evaluated in ovarian epithelial cancer (OEC) using an in vitro adenosine triphosphate tumor chemosensitivity assay (ATP-TCA). Specimens were collected from 80 patients who underwent cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissues were tested for sensitivity to paclitaxel (PTX), carboplatin (CBP), topotecan (TPT), gemcitabine (GEM), docetaxel (TXT), etoposide, bleomycin and 4-hydroperoxycyclophosphamide using ATP-TCA. The sensitivity, specificity, positive predictive value and negative predictive value for the clinical chemotherapy sensitivity of OEC were 88.6, 77.8, 83 and 84.8%, respectively. PTX demonstrated the highest sensitivity of all agents tested (82.5% in all specimens, 85.7% in recurrent specimens), followed by CBP (58.8 and 60.7%, respectively). The sensitivities to PTX and docetaxel (PIII) or low-differentiated specimens, respectively. The present study indicated that ATP-TCA is an effective method for guiding the choice of chemotherapy drugs. Notable heterogeneity of chemosensitivity was observed in the OEC specimens.

Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

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Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

2014-03-01

Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071.

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Zebisch, Matthias; Baqi, Younis; Schäfer, Petra; Müller, Christa E; Sträter, Norbert

2014-03-01

In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis. Copyright © 2014 Elsevier Inc. All rights reserved.

Protective role of hypoxia-inducible factor-1α-dependent CD39 and CD73 in fulminant acute liver failure

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Tak, Eunyoung [Asan Institute for Life Sciences and Asan-Minnesota Institute for Innovating Transplantation, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Jung, Dong-Hwan; Kim, Seok-Hwan; Park, Gil-Chun [Division of Liver Transplantation and Hepatobiliary Surgery, Asan-Minnesota Institute for Innovating Transplantation, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Jun, Dae Young; Lee, Jooyoung [Asan Institute for Life Sciences and Asan-Minnesota Institute for Innovating Transplantation, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Jung, Bo-hyun [Department of Surgery, Inje University Haeundae Paik Hospital, Inje University College of Medicine, Busan (Korea, Republic of); Kirchner, Varvara A. [Division of Transplantation, Department of Surgery and Asan-Minnesota Institute for Innovating Transplantation, University of Minnesota, Minneapolis, MN (United States); Hwang, Shin [Division of Liver Transplantation and Hepatobiliary Surgery, Asan-Minnesota Institute for Innovating Transplantation, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Song, Gi-Won, E-mail: drsong71@amc.seoul.kr [Division of Liver Transplantation and Hepatobiliary Surgery, Asan-Minnesota Institute for Innovating Transplantation, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of); Lee, Sung-Gyu [Division of Liver Transplantation and Hepatobiliary Surgery, Asan-Minnesota Institute for Innovating Transplantation, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea, Republic of)

2017-01-01

Acute liver failure (ALF) is a severe life-threatening disease which usually arises in patients with-irreversible liver illnesses. Although human ectonucleotide triphosphate diphosphohydrolase-1, E-NTPDase1 (CD39) and ecto-5′-nucleotidase, Ecto5′NTase (CD73) are known to protect tissues from ALF, the expression and function of CD39 and CD73 during ALF are currently not fully investigated. We tested whether CD39 and CD73 are upregulated by hypoxia inducible factor (HIF)-1α, and improve ischemic tolerance to ALF. To test our hypothesis, liver biopsies were obtained and we found that CD39 and CD73 mRNA and proteins from human specimens were dramatically elevated in ALF. We investigated that induction of CD39 and CD73 in ALF-related with wild type mice. In contrast, deletion of cd39 and cd73 mice has severe ALF. In this study, we concluded that CD39 and CD73 are molecular targets for the development of drugs for ALF patients care. - Highlights: • HIF-1a is stabilized during acute liver failure • Upregulation of CD39 and CD73 following acute liver failure • CD39 and CD73 are transcriptionally induced by HIF-1a • Deletion of Cd39 and CD73 aggravates murine acute liver failure • DMOG treatment induces HIF-1a stabilization, CD39 and CD73 during acute liver failure in WT mice.

Effects of 5 Thio-D-Glucose on cellular adenosine triphosphate levels and deoxyribonucleic acid rejoining in hypoxic and aerobic Chinese hamster cells

International Nuclear Information System (INIS)

Nagle, W.A.; Moss, A.J. Jr.; Roberts, H.G. Jr.; Baker, M.L.

1980-01-01

Intracellular adenosine triphosphate (ATP) levels were measured in both hypoxic and aerobic cultures of V79 Chinese hamster cells treated with 5-thio-D-glucose (5-SH-D-Glc). This glucose analog, a known inhibitor of D-glucose transport and metabolism, reduced ATP in cell cultures allowed to become hypoxic by cell metabolism, but not in aerobic cultures treated similarly. Cells depleted of ATP were unable to rejoin x-ray induced deoxyribonucleic acid (DNA) strand breaks as measured by the alkaline sucrose gradient sedimentation technique. The inference for radiation therapy is that inhibition of glucose metabolism selectively depletes energy reserves in hypoxic cells, rendering these cells more radiosensitive and leading to a more effective tumor treatment

Phosphorus Partitioning of Soybean Lines Containing Different Mutant Alleles of Two Soybean Seed-Specific Adenosine Triphosphate-Binding Cassette Phytic Acid Transporter Paralogs

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Jason D. Gillman

2013-03-01

Full Text Available Seed phytate is a repository of P and minerals in soybean [ (L. Merr.] seeds that limits P and mineral bioavailability for monogastric animals (e.g., humans, swine [], and poultry [especially chicken, ] due to insufficient digestive tract phytase activity. We previously identified epistatic recessive mutations affecting two paralogous adenosine triphosphate-binding cassette phytic acid transporter genes (one a nonsense mutation in and the other a missense mutation in as the molecular genetic basis in the ethyl methanesulfonate (EMS-induced mutant low phytate soybean line M153. An additional mutant low phytate line, M766, contained one single nucleotide polymorphism within the ninth intron of the locus as well as a nonsense mutation in . The objectives of this research were to clarify the genetics underlying the low phytate phenotype in line M766 and to determine P partitioning in new combinations of mutant alleles from M766 and M153. Inheritance of nonsense alleles affecting both ( genes (one from M153 and one from M766 led to the production of viable seeds that contained transgressive reductions in total seed phytate and significantly higher levels of inorganic phosphate than has been reported for nontransgenic soybean material and will allow efficient molecular selection of soybeans with even greater reductions of phytate for improved quality soybean meal.

Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

Science.gov (United States)

Lin, Jia-Hui; Yang, Ya-Chun; Shih, Ya-Chen; Hung, Szu-Ying; Lu, Chi-Yu; Tseng, Wei-Lung

2016-03-15

Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

Energy Technology Data Exchange (ETDEWEB)

Shyy, Y.J.; Tian, G.; Tsai, M.D.

1987-10-06

Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

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Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

2010-07-01

Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

Oral sucrose for heel lance increases adenosine triphosphate use and oxidative stress in preterm neonates.

Science.gov (United States)

Asmerom, Yayesh; Slater, Laurel; Boskovic, Danilo S; Bahjri, Khaled; Holden, Megan S; Phillips, Raylene; Deming, Douglas; Ashwal, Stephen; Fayard, Elba; Angeles, Danilyn M

2013-07-01

To examine the effects of sucrose on pain and biochemical markers of adenosine triphosphate (ATP) degradation and oxidative stress in preterm neonates experiencing a clinically required heel lance. Preterm neonates that met study criteria (n = 131) were randomized into 3 groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking; and (3) heel lance treated with sucrose and non-nutritive sucking. Plasma markers of ATP degradation (hypoxanthine, xanthine, and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured with the Premature Infant Pain Profile. Data were analyzed by the use of repeated-measures ANOVA and Spearman rho. We found significant increases in plasma hypoxanthine and uric acid over time in neonates who received sucrose. We also found a significant negative correlation between pain scores and plasma allantoin concentration in a subgroup of neonates who received sucrose. A single dose of oral sucrose, given before heel lance, significantly increased ATP use and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effects of repeated sucrose administration on ATP degradation, oxidative stress, and cell injury. Copyright © 2013 Mosby, Inc. All rights reserved.

Prolonged Atrioventricular Block and Ventricular Standstill Following Adenosine Triphosphate Injection in a Patient Taking Dipyridamole and Antiarrhythmic Agents: A Case Report

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Kotaro Oe, MD

2009-01-01

Full Text Available An 83-year-old woman was admitted to our hospital because of palpitation. She had hypertension and paroxysmal atrial fibrillation, treated with digoxin and cibenzoline, and took dipyridamole for microalbuminuria. Before admission, she had taken pilsicainide pills in addition. On admission, electrocardiogram showed regular tachycardia with mildly prolonged QRS width. For the purpose of terminating tachycardia, 10 mg of adenosine triphosphate (ATP was rapidly injected. About 20 sec later, atrioventricular block and ventricular standstill occurred. She presented loss of consciousness and convulsion, and chest compression was performed. About 30 sec later, the QRS complex reappeared, and she became alert. Serum concentration of digoxin, cibenzoline and pilsicainide was within therapeutic level, respectively. We should be cautious in using ATP for a patient taking dipyridamole and antiarrhythmic agents.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

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Moulin, Morgane; Alguacil, Javier; Gu, Siyi; Mehtougui, Asmaa; Adams, Erin J; Peyrottes, Suzanne; Champagne, Eric

2017-12-01

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Dapper1 attenuates hepatic gluconeogenesis and lipogenesis by activating PI3K/Akt signaling.

Science.gov (United States)

Kuang, Jian-Ren; Zhang, Zhi-Hui; Leng, Wei-Ling; Lei, Xiao-Tian; Liang, Zi-Wen

2017-05-15

Studies have shown that hepatic insulin resistance, a disorder of glucose and lipid metabolism, plays a vital role in type 2 diabetes (T2D). To clarify the function of Dapper1 in glucose and lipid metabolism in the liver, we investigated the relationships between Dapper1 and adenosine triphosphate (ATP)- and Ca 2+ -mediated activation of PI3K/Akt. We observed a reduction in hepatic Dapper1 in db/db (mice that are homozygous for a spontaneous diabetes mutation) and HFD-induced diabetic mice with T2D. Hepatic overexpression of Dapper1 improved hyperglycemia, insulin resistance, and fatty liver. It also increased Akt (pAkt) signaling and repressed both gluconeogenesis and lipogenesis. Conversely, Ad-shDapper1-induced knockdown of hepatic Dapper1 promoted gluconeogenesis and lipogenesis. Furthermore, Dapper1 activated PI3K p110α/Akt in an insulin-independent manner by inducing ATP production and secretion in vitro. Blockade of P2 ATP receptors, the downstream phospholipase C (PLC), or the inositol triphosphate receptor (IP3R all reduced the Dapper1-induced increase in cytosolic free calcium and Dapper1-mediated PI3K/Akt activation, as did removal of calcium in the medium. In conclusion, Dapper1 attenuates hepatic gluconeogenesis and lipogenesis in T2D. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and characterization of a gene encoding Rac/Rop-like monomeric guanosine 5'-triphosphate-binding protein from Scoparia dulcis.

Science.gov (United States)

Mitamura, Toshiaki; Shite, Masato; Yamamura, Yoshimi; Kurosaki, Fumiya

2009-06-01

A cDNA clone, designated Sd-racrop (969 bp), was isolated from seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 197 amino acid residues with high homology to Rac/Rop small guanosine 5'-triphosphate-binding proteins from various plant sources. In Southern hybridization analysis, the restriction digests prepared from genomic DNA of S. dulcis showed a main signal together with a few weakly hybridized bands. The transcriptional level of Sd-racrop showed a transient decrease by exposure of the leaf tissues of S. dulcis to the ethylene-generating reagent 2-chloroethylphosphonic acid. However, an appreciable increase in gene expression was reproducibly observed upon treatment of the plant with methyl jasmonate. These results suggest that the Sd-racrop product plays roles in ethylene- and methyl jasmonate-induced responses of S. dulcis accompanying the change in the transcriptional level, however, the cellular events mediated by this protein toward these external stimuli would be regulated by various mechanisms.

Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

Science.gov (United States)

Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

2008-11-01

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice.

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Bayliss, Jill; Delarosa, Sara; Wu, Jianfeng; Peterson, Jonathan R; Eboda, Oluwatobi N; Su, Grace L; Hemmila, Mark; Krebsbach, Paul H; Cederna, Paul S; Wang, Stewart C; Xi, Chuanwu; Levi, Benjamin

2014-01-01

Extracellular adenosine triphosphate (ATP), present in thermally injured tissue, modulates the inflammatory response and causes significant tissue damage. The authors hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site. Mice were subjected to 30% TBSA partial-thickness scald burn by dorsal skin immersion in a water bath at 60 or 20°C (nonburn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain, elastase, and Ki-67 staining. Tumor necrosis factor (TNF)-α and interferon (IFN)-β expression were measured through quantitative real-time polymerase chain reaction. At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (P burn group at 24 hours and 5 days. TNF-α and IFN-β expression at 24 hours in the apyrase group was lower than in the burn group (P burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially because of TNF-α and IFN-β signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of health claims related to sugar-free chewing gum with pyro- and triphosphates and reduction of calculus formation (ID 1309) pursuant to Article 13(1) of Regulation (EC) No 1924/2006

DEFF Research Database (Denmark)

Tetens, Inge

calculus/tartar formation, gums health”. The target population is assumed to be the general population. The Panel considers that reduction of calculus formation at sites which are most important for dental health is a beneficial physiological effect. No human studies have been provided from which...... conclusions could be drawn for the scientific substantiation of a claim on the use of sugar-free chewing gum with pyro- and triphosphates and the reduction of calculus formation at sites which are most important for dental health (e.g. gingival margin or between teeth). On the basis of the data presented......, the Panel concludes that a cause and effect relationship has not been established between the use of sugar-free chewing gum with pyro- and triphosphates and reduction of calculus formation at sites which are most important for dental health....

Colorimetric sensor for triphosphates and their application as a viable staining agent for prokaryotes and eukaryotes.

Science.gov (United States)

Ghosh, Amrita; Shrivastav, Anupama; Jose, D Amilan; Mishra, Sanjiv K; Chandrakanth, C K; Mishra, Sandhya; Das, Amitava

2008-07-15

The chromogenic complex 1 x Zn (where 1 is (E)-4-(4-dimethylamino-phenylazo)-N,N-bispyridin-2-ylmethyl-benzenesulfonamide) showed high affinity toward the phosphate ion in tetrabutylammonium phosphate in acetonitrile solution and could preferentially bind to adenosine triphosphate (ATP) in aqueous solution at physiological pH. This binding caused a visual change in color, whereas no such change was noticed with other related anions (adenosine monophosphate, adenosine diphosphate, pyrophosphate, and phosphate) of biological significance. Thus, 1 x Zn could be used as a staining agent for different biological cells through binding to the ATP, generated in situ by the mitochondria (in eukaryotes). For prokaryotes (bacteria) the cell membrane takes care of the cells' energy conversion, since they lack mitochondria. ATP is produced in their unique cell structure on the cell membrane, which is not found in any eukaryotes. These stained cells could be viewed with normal light microscopy. This reagent could even be used for distinguishing the gram-positive and the gram-negative bacteria (prokaryotes). This dye was found to be nonlipophilic in nature and nontoxic to living microbes (eukaryotes and prokaryotes). Further, stained cells were found to grow in their respective media, and this confirmed the maintenance of viability of the microbes even after staining, unlike with many other dyes available commercially.

Schistosome tegumental ecto-apyrase (SmATPDase1 degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

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Akram A. Da’dara

2014-03-01

Full Text Available Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP. Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP, phosphodiesterase (SmNPP-5 and ATP diphosphohydrolase (SmATPDase1. In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

Science.gov (United States)

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity

Energy Technology Data Exchange (ETDEWEB)

Hung, Szu-Ying; Shih, Ya-Chen [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); Tseng, Wei-Lung, E-mail: tsengwl@mail.nsysu.edu.tw [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan (China); Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Taiwan (China); Center for Stem Cell Research, Kaohsiung Medical University, Taiwan (China)

2015-02-01

Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity

International Nuclear Information System (INIS)

Hung, Szu-Ying; Shih, Ya-Chen; Tseng, Wei-Lung

2015-01-01

Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

Actions of p-synephrine on hepatic enzyme activities linked to carbohydrate metabolism and ATP levels in vivo and in the perfused rat liver.

Science.gov (United States)

Maldonado, Marcos Rodrigues; Bracht, Lívia; de Sá-Nakanishi, Anacharis Babeto; Corrêa, Rúbia Carvalho Gomes; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

2018-01-01

p-Synephrine is one of the main active components of the fruit of Citrus aurantium (bitter orange). Extracts of the bitter orange and other preparations containing p-synephrine have been used worldwide to promote weight loss and for sports performance. The purpose of the study was to measure the action of p-synephrine on hepatic enzyme activities linked to carbohydrate and energy metabolism and the levels of adenine mononucleotides. Enzymes and adenine mononucleotides were measured in the isolated perfused rat liver and in vivo after oral administration of the drug (50 and 300 mg/kg) by using standard techniques. p-Synephrine increased the activity of glycogen phosphorylase in vivo and in the perfused liver. It decreased, however, the activities of pyruvate kinase and pyruvate dehydrogenase also in vivo and in the perfused liver. p-Synephrine increased the hepatic pools of adenosine diphosphate and adenosine triphosphate. Stimulation of glycogen phosphorylase is consistent with the reported increased glycogenolysis in the perfused liver and increased glycemia in rats. The decrease in the pyruvate dehydrogenase activity indicates that p-synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. The capability of increasing the adenosine triphosphate-adenosine diphosphate pool indicates a beneficial effect of p-synephrine on the cellular energetics. Copyright © 2017 John Wiley & Sons, Ltd.

A quantitative analysis of the effects of 2,3-diphosphoglycerate, adenosine triphosphate and inositol hexaphosphate on the oxygen dissociation curve of human haemoglobin.

Science.gov (United States)

Goodford, P J; St-Louis, J; Wootton, R

1978-01-01

1. Oxygen dissociation curves have been measured for human haemoglobin solutions with different concentrations of the allosteric effectors 2,3-diphosphoglycerate, adenosine triphosphate and inositol hexaphosphate. 2. Each effector produces a concentration dependent right shift of the oxygen dissociation curve, but a point is reached where the shift is maximal and increasing the effector concentration has no further effect. 3. Mathematical models based on the Monod, Wyman & Changeux (1965) treatment of allosteric proteins have been fitted to the data. For each compound the simple two-state model and its extension to take account of subunit inequivalence were shown to be inadequate, and a better fit was obtained by allowing the effector to lower the oxygen affinity of the deoxy conformational state as well as binding preferentially to this conformation. PMID:722582

Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

DEFF Research Database (Denmark)

Munch-Petersen, Birgitte

2010-01-01

The central enzyme on the de novo pathway for synthesis of DNA precursors, the deoxyribonucleoside triphosphates, is ribonucleotide reductase (RNR). Deoxythymidine triphosphate (dTTP) has a key role in control of RNR activity shifting the specificity from pyrimidine to purine nucleotide reduction...

Use of adenosine triphosphate to audit reprocessing of flexible endoscopes with an elevator mechanism.

Science.gov (United States)

Quan, Erik; Mahmood, Rizwan; Naik, Amar; Sargon, Peter; Shastri, Nikhil; Venu, Mukund; Parada, Jorge P; Gupta, Neil

2018-05-21

There have been reported outbreaks of carbapenem-resistant Enterobacteriaceae infections linked to endoscopes with elevator mechanisms. Adenosine triphosphate (ATP) testing has been used as a marker for bioburden and monitoring manual cleaning for flexible endoscopes with and without an elevator mechanism. The objective of this study was to determine whether routine ATP testing could identify areas of improvement in cleaning of endoscopes with an elevator mechanism. ATP testing after manual cleaning of TJF-Q180V duodenoscopes and GF-UCT180 linear echoendoscopes (Olympus America Inc, Center Valley, PA) was implemented. Samples were tested from the distal end, the elevator mechanism, and water flushed through the lumen of the biopsy channel. Data were recorded and compared by time point, test point, and reprocessing technician. Overall failure rate was 6.99% (295 out of 4,219). The highest percentage of failed ATP tests (17.05%) was reported in the first quarter of routine testing, with an overall decrease in rates over time. The elevator mechanism and working channel lumen had higher failure rates than the distal end. Quality of manual cleaning between reprocessing technicians showed variation. ATP testing is effective in identifying residual organic material and improving quality of manual cleaning of endoscopes with an elevator mechanism. Cleaning efficacy is influenced by reprocessing technicians and location tested on the endoscope. Close attention to the working channel and elevator mechanism during manual cleaning is warranted. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

ABC gene expression profiles have clinical importance and possibly form a new hallmark of cancer.

Science.gov (United States)

Dvorak, Pavel; Pesta, Martin; Soucek, Pavel

2017-05-01

Adenosine triphosphate-binding cassette proteins constitute a large family of active transporters through extracellular and intracellular membranes. Increased drug efflux based on adenosine triphosphate-binding cassette protein activity is related to the development of cancer cell chemoresistance. Several articles have focused on adenosine triphosphate-binding cassette gene expression profiles (signatures), based on the expression of all 49 human adenosine triphosphate-binding cassette genes, in individual tumor types and reported connections to established clinicopathological features. The aim of this study was to test our theory about the existence of adenosine triphosphate-binding cassette gene expression profiles common to multiple types of tumors, which may modify tumor progression and provide clinically relevant information. Such general adenosine triphosphate-binding cassette profiles could constitute a new attribute of carcinogenesis. Our combined cohort consisted of tissues from 151 cancer patients-breast, colorectal, and pancreatic carcinomas. Standard protocols for RNA isolation and quantitative real-time polymerase chain reaction were followed. Gene expression data from individual tumor types as well as a merged tumor dataset were analyzed by bioinformatics tools. Several general adenosine triphosphate-binding cassette profiles, with differences in gene functions, were established and shown to have significant relations to clinicopathological features such as tumor size, histological grade, or clinical stage. Genes ABCC7, A3, A8, A12, and C8 prevailed among the most upregulated or downregulated ones. In conclusion, the results supported our theory about general adenosine triphosphate-binding cassette gene expression profiles and their importance for cancer on clinical as well as research levels. The presence of ABCC7 (official symbol CFTR) among the genes with key roles in the profiles supports the emerging evidence about its crucial role in various

Probing the communication of deoxythymidine triphosphate in HIV-1 reverse transcriptase by communication maps and interaction energy studies.

Science.gov (United States)

Gnanasekaran, Ramachandran

2017-11-08

We calculate communication maps for HIV-1 Reverse Transcriptase (RT) to elucidate energy transfer pathways between deoxythymidine triphosphate (dTTP) and other parts of the protein. This approach locates energy transport channels from the dTTP to remote regions of the protein via residues and water molecules. We examine the water dynamics near the catalytic site of HIV-1 RT by molecular dynamics (MD) simulations. We find that, within the catalytic site, the relaxation of water molecules is similar to that of the hydration water molecules present in other proteins and the relaxation time scale is fast enough to transport energy and helps in communication between dTTP and other residues in the system. To quantify energy transfer, we also calculate the interaction energies of dTTP, 2Mg 2+ , doxy-guanosine nucleotide (DG22) with their surrounding residues by using the B3LYP-D3 method. The results, from classical vibrational energy diffusivity and QM interaction energy, are complementary to identify the important residues involved in the process of polymerization. The positive and negative interactions by dTTP with different types of residues in the catalytic region make the residues transfer energy through vibrational communication.

A novel fluorescent biosensor for Adenosine Triphosphate detection based on the polydopamine nanospheres integrating with enzymatic recycling amplification.

Science.gov (United States)

Ji, Xiaoting; Yi, Bingqing; Xu, Yujuan; Zhao, Yanan; Zhong, Hua; Ding, Caifeng

2017-07-01

Based on the protective performance of polydopamine nanospheres (PDANSs) for DNA against nuclease digestion and the specific recognition characteristic of aptamer, we have developed an enzymatic recycling signal amplification method for highly sensitive and selective detection of adenosine triphosphate (ATP). Fluorescence measurements were carried out to verify the DNA polymerase and exonuclease III (Exo III) assisted target recycling process and fluorescence signal amplification. In the absence of the ATP, initially, the signal DNA-PDANSs complex was in the "off" state due to the efficient fluorescence quenching of 6-carboxyfluorescein (FAM) adjacent to the surface of PDANSs. Due to the binding of the aptamer by ATP, it trigger DNA polymerase and Exo III assisted target recycling process by the product of release, the complex would change into the "on" state as a result of the dissociation of the FAM from the surface of PDANSs, thus providing greatly enhanced fluorescence emission intensity. The method allows quantitative detection of ATP in the range of 20-600nM with a detection limit of 8.32nM. This biosensor requires no complex operations, and is a new high efficiency method for ATP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy.

Science.gov (United States)

Ambruso, D R; Hawkins, B; Johnson, D L; Fritzberg, A R; Klingensmith, W C; McCabe, E R

1986-06-01

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.

Effects of caffeine on fractional flow reserve values measured using intravenous adenosine triphosphate.

Science.gov (United States)

Nakayama, Masafumi; Chikamori, Taishiro; Uchiyama, Takashi; Kimura, Yo; Hijikata, Nobuhiro; Ito, Ryosuke; Yuhara, Mikio; Sato, Hideaki; Kobori, Yuichi; Yamashina, Akira

2018-04-01

We investigated the effects of caffeine intake on fractional flow reserve (FFR) values measured using intravenous adenosine triphosphate (ATP) before cardiac catheterization. Caffeine is a competitive antagonist for adenosine receptors; however, it is unclear whether this antagonism affects FFR values. Patients were evenly randomized into 2 groups preceding the FFR study. In the caffeine group (n = 15), participants were given coffee containing 222 mg of caffeine 2 h before the catheterization. In the non-caffeine group (n = 15), participants were instructed not to take any caffeine-containing drinks or foods for at least 12 h before the catheterization. FFR was performed in patients with more than intermediate coronary stenosis using the intravenous infusion of ATP at 140 μg/kg/min (normal dose) and 170 μg/kg/min (high dose), and the intracoronary infusion of papaverine. FFR was followed for 30 s after maximal hyperemia. In the non-caffeine group, the FFR values measured with ATP infusion were not significantly different from those measured with papaverine infusion. However, in the caffeine group, the FFR values were significantly higher after ATP infusion than after papaverine infusion (P = 0.002 and P = 0.007, at normal and high dose ATP vs. papaverine, respectively). FFR values with ATP infusion were significantly increased 30 s after maximal hyperemia (P = 0.001 and P < 0.001 for normal and high dose ATP, respectively). The stability of the FFR values using papaverine showed no significant difference between the 2 groups. Caffeine intake before the FFR study affected FFR values and their stability. These effects could not be reversed by an increased ATP dose.

PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

Science.gov (United States)

Magnard, Jean-Louis; Roccia, Aymeric; Caissard, Jean-Claude; Vergne, Philippe; Sun, Pulu; Hecquet, Romain; Dubois, Annick; Hibrand-Saint Oyant, Laurence; Jullien, Frédéric; Nicolè, Florence; Raymond, Olivier; Huguet, Stéphanie; Baltenweck, Raymonde; Meyer, Sophie; Claudel, Patricia; Jeauffre, Julien; Rohmer, Michel; Foucher, Fabrice; Hugueney, Philippe; Bendahmane, Mohammed; Baudino, Sylvie

2015-07-03

The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta. Copyright © 2015, American Association for the Advancement of Science.

Regulation of ROCK Activity in Cancer

Science.gov (United States)

Morgan-Fisher, Marie; Wewer, Ulla M.

2013-01-01

Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)–loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer. PMID:23204112

Monitoring Biological Activity at Geothermal Power Plants

Energy Technology Data Exchange (ETDEWEB)

Peter Pryfogle

2005-09-01

The economic impact of microbial growth in geothermal power plants has been estimated to be as high as $500,000 annually for a 100 MWe plant. Many methods are available to monitor biological activity at these facilities; however, very few plants have any on-line monitoring program in place. Metal coupon, selective culturing (MPN), total organic carbon (TOC), adenosine triphosphate (ATP), respirometry, phospholipid fatty acid (PLFA), and denaturing gradient gel electrophoresis (DGGE) characterizations have been conducted using water samples collected from geothermal plants located in California and Utah. In addition, the on-line performance of a commercial electrochemical monitor, the BIoGEORGE?, has been evaluated during extended deployments at geothermal facilities. This report provides a review of these techniques, presents data on their application from laboratory and field studies, and discusses their value in characterizing and monitoring biological activities at geothermal power plants.

Electrochemical oxidation of adenosine-5 Prime -triphosphate on a chitosan-graphene composite modified carbon ionic liquid electrode and its determination

Energy Technology Data Exchange (ETDEWEB)

Sun Wei, E-mail: swyy26@hotmail.com [College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158 (China); College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China); Liu Jun; Wang Xiuzhen; Li Tongtong; Li Guangjiu; Wu Jie [College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China); Zhang Liqi [State Key Laboratory of Coal Combustion, Huazhong University of Science and Technology, Wuhan 430074 (China)

2012-10-01

In this paper a new electrochemical method was proposed for the determination of adenosine-5 Prime -triphosphate (ATP) based on a chitosan (CTS) and graphene (GR) composite film modified carbon ionic liquid electrode (CTS-GR/CILE). CILE was fabricated by using ionic liquid 1-butyl-3-methylimidazolium dihydrogen phosphate ([BMIM]H{sub 2}PO{sub 4}) as the binder, which was further modified by GR and CTS composite. The modified electrode exhibited an excellent electrocatalytic activity toward the oxidation of ATP with the increase of the oxidation peak current and the decrease of the oxidation peak potential. The electrochemical parameters of ATP on CTS-GR/CILE were calculated with the electron transfer coefficient ({alpha}) as 0.329, the electron transfer number (n) as 2.15, the apparent heterogeneous electron transfer rate constant (ks) as 3.705 Multiplication-Sign 10{sup -5} s{sup -1} and the surface coverage ({Gamma}{sub T}) as 9.33 Multiplication-Sign 10{sup -10} mol cm{sup -2}. Under the optimal conditions the oxidation peak current was proportional to ATP concentration in the range from 1.0 Multiplication-Sign 10{sup -6} to 1.0 Multiplication-Sign 10{sup -3} M with the detection limit of 0.311 {mu}M (S/N = 3). The proposed electrode showed excellent reproducibility, stability, anti-interference ability and further successfully applied to the ATP injection sample detection. - Highlights: Black-Right-Pointing-Pointer Ionic liquid [BMIM]H{sub 2}PO{sub 4} based carbon ionic liquid electrode (CILE) was prepared. Black-Right-Pointing-Pointer Graphene modified CILE was fabricated for the sensitive electrochemical detection of ATP. Black-Right-Pointing-Pointer Good electrocatalytic ability to the ATP oxidation was achieved. Black-Right-Pointing-Pointer Detection of 5 Prime -ATP in commercial injection samples with satisfactory results.

The effect of experimental gastric dilatation-volvulus on adenosine triphosphate content and conductance of the canine gastric and jejunal mucosa.

Science.gov (United States)

Peycke, Laura E; Hosgood, Giselle; Davidson, Jacqueline R; Tetens, Joanne; Taylor, H Wayne

2005-07-01

The objective of this study was to determine if experimental gastric dilatation volvulus (GDV) would decrease adenosine triphosphate (ATP) concentration and increase membrane conductance of the canine gastric and jejunal mucosa. Male dogs (n = 15) weighing between 20 and 30 kg were used. Dogs were randomly assigned to 1 of 3 equal groups: Group 1 was control, group 2 was GDV, and group 3 was ischemia. All dogs were anesthetized for 210 min. Group 1 had no manipulation. Group 2 had GDV experimentally induced for 120 min followed by decompression, derotation, and reperfusion for 90 min. Group 3 had GDV experimentally induced for 210 min. Gastric (fundus and pylorus) and jejunal tissue was taken at 0, 120, and 210 min from all of the dogs. Tissue was analyzed for ATP concentration, mucosal conductance, and microscopic changes. The ATP concentration in the fundus did not change significantly from baseline in group 2, but decreased significantly below baseline at 210 min in group 3. The ATP concentration in the jejunum decreased significantly below baseline in groups 2 and 3 at 120 min, remaining significantly decreased in group 3 but returning to baseline at 210 min in group 2. Mucosal conductance of the fundus did not change significantly in any dog. Mucosal conductance of the jejunum increased at 120 min in groups 2 and 3, and became significantly increased above baseline at 210 min. The jejunal mucosa showed more profound cellular changes than the gastric mucosa. The jejunum showed substantial decreases in ATP concentration with an increase in mucosal conductance, suggesting cell membrane dysfunction. Dogs sustaining a GDV are likely to have a change in the activity of mucosal cells in the jejunum, which may be important in the pathophysiology of GDV.

Antifouling aptasensor for the detection of adenosine triphosphate in biological media based on mixed self-assembled aptamer and zwitterionic peptide.

Science.gov (United States)

Wang, Guixiang; Su, Xiaoli; Xu, Qingjun; Xu, Guiyun; Lin, Jiehua; Luo, Xiliang

2018-03-15

Direct detection of targets in complex biological media with conventional biosensors is an enormous challenge due to the nonspecific adsorption and severe biofouling. In this work, a facile strategy for sensitive and low fouling detection of adenosine triphosphate (ATP) is developed through the construction of a mixed self-assembled biosensing interface, which was composed of zwitterionic peptide (antifouling material) and ATP aptamer (bio-recognition element). The peptide and aptamer (both containing thiol groups) were simultaneously self-assembled onto gold electrode surface electrodeposited with gold nanoparticles. The developed aptasensor possessed high selectivity and sensitivity for ATP, and it showed a wide linear response range towards ATP from 0.1pM to 5nM. Owing to the presence of peptide with excellent antifouling property in the biosensing interface, the aptasensor can detect ATP in complex biological media with remarkably reduced biofouling or nonspecific adsorption effect. Moreover, it can directly detect ATP in 1% human whole blood without suffering from any significant interference, indicating its great potential for practical assaying of ATP in biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Enzyme-free and label-free ultrasensitive electrochemical detection of DNA and adenosine triphosphate by dendritic DNA concatamer-based signal amplification.

Science.gov (United States)

Liu, Shufeng; Lin, Ying; Liu, Tao; Cheng, Chuanbin; Wei, Wenji; Wang, Li; Li, Feng

2014-06-15

Hybridization chain reaction (HCR) strategy has been well developed for the fabrication of various biosensing platforms for signal amplification. Herein, a novel enzyme-free and label-free ultrasensitive electrochemical DNA biosensing platform for the detection of target DNA and adenosine triphosphate (ATP) was firstly proposed, in which three auxiliary DNA probes were ingeniously designed to construct the dendritic DNA concatamer via HCR strategy and used as hexaammineruthenium(III) chloride (RuHex) carrier for signal amplification. With the developed dendritic DNA concatamer-based signal amplification strategy, the DNA biosensor could achieve an ultrasensitive electrochemical detection of DNA and ATP with a superior detection limit as low as 5 aM and 20 fM, respectively, and also demonstrate a high selectivity for DNA and ATP detection. The currently proposed dendritic DNA concatamer opens a promising direction to construct ultrasensitive DNA biosensing platform for biomolecular detection in bioanalysis and clinical biomedicine, which offers the distinct advantages of simplicity and cost efficiency owing to no need of any kind of enzyme, chemical modification or labeling. Copyright © 2014 Elsevier B.V. All rights reserved.

Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers.

Science.gov (United States)

Ratajczak, Katarzyna; Stobiecka, Magdalena

2017-07-20

The interactions of fluorescent probes and biomolecules with nanocarriers are of key importance to the emerging targeted drug delivery systems. Graphene oxide nanosheets (GONs) as the nanocarriers offer biocompatibility and robust drug binding capacity. The interactions of GONs with fluorophores lead to strong fluorescence quenching, which may interfere with fluorescence bioimaging and biodetection. Herein, we report on the interactions and energy transfers in a model ternary system: GONs-FITC-ATP, where FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'-triphosphate). We have found that FITC fluorescence is considerably quenched by ATP (the quenching constant K SV = 113 ± 22 M -1 ). The temperature coefficient of K SV is positive (α T = 4.15 M -1 deg -1 ). The detailed analysis of a model for internal self-quenching of FITC indicates that the temperature dependence of the net quenching efficiency η for the FITC-ATP pair is dominated by FITC internal self-quenching modes with their contribution estimated at 79%. The quenching of FITC by GONs is much stronger (K SV = 598 ± 29 M -1 ) than that of FITC-ATP and is associated with the formation of supramolecular assemblies bound with hydrogen bonding and π-π stacking interactions. For the analysis of the complex behavior of the ternary system GONs-FITC-ATP, a model of chemisorption of ATP on GONs, with partial blocking of FITC quenching, has been developed. Our results indicate that ATP acts as a moderator for FITC quenching by GONs. The interactions between ATP, FITC, and GONs have been corroborated using molecular dynamics and quantum mechanical calculations.

Visual and surface plasmon resonance sensor for zirconium based on zirconium-induced aggregation of adenosine triphosphate-stabilized gold nanoparticles.

Science.gov (United States)

Qi, Wenjing; Zhao, Jianming; Zhang, Wei; Liu, Zhongyuan; Xu, Min; Anjum, Saima; Majeed, Saadat; Xu, Guobao

2013-07-17

Owing to its high affinity with phosphate, Zr(IV) can induce the aggregation of adenosine 5'-triphosphate (ATP)-stabilized AuNPs, leading to the change of surface plasmon resonance (SPR) absorption spectra and color of ATP-stabilized AuNP solutions. Based on these phenomena, visual and SPR sensors for Zr(IV) have been developed for the first time. The A(660 nm)/A(518 nm) values of ATP-stabilized AuNPs in SPR absorption spectra increase linearly with the concentrations of Zr(IV) from 0.5 μM to 100 μM (r=0.9971) with a detection limit of 95 nM. A visual Zr(IV) detection is achieved with a detection limit of 30 μM. The sensor shows excellent selectivity against other metal ions, such as Cu(2+), Fe(3+), Cd(2+), and Pb(2+). The recoveries for the detection of 5 μM, 10 μM, 25 μM and 75 μM Zr(IV) in lake water samples are 96.0%, 97.0%, 95.6% and 102.4%, respectively. The recoveries of the proposed SPR method are comparable with those of ICP-OES method. Copyright © 2013 Elsevier B.V. All rights reserved.

[Adenylate cyclase from rabbit heart: substrate binding site].

Science.gov (United States)

Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

1981-08-01

The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin-Al3+ complex and its analytical application.

Science.gov (United States)

Kamruzzaman, Mohammad; Faruqui, A Nayeem; Hossain, Mohammed Ifteker; Lee, Sang Hak

2015-11-01

A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)-aluminium (III) (Al(3+) ) complex and ATP and studied using UV-visible and fluorescence spectroscopy. Weak luminescence spectra of Al(3+) were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al(3+) ion. It was observed that the FL emission spectrum of GFLX-Al(3+) was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10(-10) - 1.0 × 10(-8) mol L(-1) with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10(-11) mol L(-1) for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10(-8) mol L(-1) ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd.

Damage to adenosine-triphosphate induced by monochromatic X rays around the K shell absorption edge of phosphorus

International Nuclear Information System (INIS)

Watanabe, Ritsuko; Ishikawa, Mitsuo; Takakura, Kaoru; Kobayashi, Katsumi

1992-01-01

Adenosine-triphosphate (ATP) is well known to have an important role in the energy metabolism in biological systems. The purpose of this study is to clarify the radiation effects on ATP specific to inner shell ionization. ATP, in concentrated aqueous solution, was irradiated with monochromatic X rays having energies of the resonance absorption peak of the phosphorus K shell, 2.153 keV, and slightly below and above the peak, 2.145 keV and 2.160 keV, selected from synchrotron radiation. Adenine, Adenosine 5'monophosphate (5'AMP) and Adenosine 5'diphosphate (5'ADP) were obtained as radioproducts by the method of high performance liquid chromatography (HPLC). G values of these products were calculated on the basis of the absorbed energy. When the ATP solution of 0.282 mol/l was irradiated with 2.160 keV X rays which can ionize the K shell of phosphorus, G values of Adenine, 5'AMP and 5'ADP were estimated to be 1.4, 0.40 and 0.46, respectively. These values were respectively 1.3, 2.9 and 3.8 times higher than those obtained upon irradiation with 2.146 keV X rays which cannot ionize the K shell of phosphorus. These energy dependent enhancements may reflect the difference in energy absorption processes, especially the Auger cascade in phosphorus may be suspected to play an important role in these enhancements

Kinetic, spectroscopic and chemical modification study of iron release from transferrin; iron(III) complexation to adenosine triphosphate

International Nuclear Information System (INIS)

Thompson, C.P.

1985-01-01

Amino acids other than those that serve as ligands have been found to influence the chemical properties of transferrin iron. The catalytic ability of pyrophosphate to mediate transferrin iron release to a terminal acceptor is largely quenched by modification non-liganded histine groups on the protein. The first order rate constants of iron release for several partially histidine modified protein samples were measured. A statistical method was employed to establish that one non-liganded histidine per metal binding domain was responsible for the reduction in rate constant. These results imply that the iron mediated chelator, pyrophosphate, binds directly to a histidine residue on the protein during the iron release process. EPR spectroscopic results are consistent with this interpretation. Kinetic and amino acid sequence studies of ovotransferrin and lactoferrin, in addition to human serum transferrin, have allowed the tentative assignment of His-207 in the N-terminal domain and His-535 in the C-terminal domain as the groups responsible for the reduction in rate of iron release. The above concepts have been extended to lysine modified transferrin. Complexation of iron(II) to adenosine triphosphate (ATP) was also studied to gain insight into the nature of iron-ATP species present at physiological pH. 31 P NMR spectra are observed when ATP is presented in large excess

Clinical characteristics in patients showing ischemic electrocardiographic changes during adenosine triphosphate loading single-photon emission computed tomography

International Nuclear Information System (INIS)

Ohtaki, Yuka; Chikamori, Taishiro; Hida, Satoshi; Tanaka, Hirokazu; Igarashi, Yuko; Hatano, Tsuguhisa; Usui, Yasuhiro; Miyagi, Manabu; Yamashina, Akira

2010-01-01

Although ischemic electrocardiographic (ECG) changes during dipyridamole or adenosine infusion have been reported as a marker for severe coronary artery disease (CAD), few studies have focused on ST-segment changes with adenosine triphosphate (ATP)-loading myocardial single-photon emission computed tomography (SPECT). Between January 2003 and August 2008, 4650 consecutive patients underwent ATP-loading SPECT. After 1412 patients with left bundle branch block, pacemaker rhythm, or previous coronary revascularization were excluded, 16 out of 3238 patients (0.5%) showed ischemic ST-segment depression during ATP-loading myocardial SPECT. They were aged 67±11 years; 10 were men and 6 women. Of these patients, 8 demonstrated perfusion abnormalities, whereas the remaining 8 showed normal myocardial perfusion imaging. In 6 of the 8 patients with abnormal SPECT, coronary angiography was performed, revealing left main trunk disease in 1 patient, 3-vessel disease in 4, 1-vessel disease with proximal left ascending artery occlusion in 1, and an insignificant lesion in 1. By contrast, no major cardiac event was observed in the 8 patients with normal SPECT during follow-up for an average of 2 years. The prevalence of ischemic ST-segment changes during ATP loading is very rare. However, this finding should be taken into account since almost half of the patients, particularly those with perfusion abnormalities, may have severe CAD which requires coronary revascularization. (author)

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Science.gov (United States)

Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

2009-01-01

Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

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Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

2014-07-04

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. Copyright © 2014, American Association for the Advancement of Science.

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.

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Ren, Wenwen; Lewandowski, Brian C; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A; Margolskee, Robert F; Jiang, Peihua

2014-11-18

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors.

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Hassan, Hammad Ali; Rani, Sadaf; Fatima, Tabeer; Kiani, Farooq Ahmad; Fischer, Stefan

2017-11-01

Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the O a H bond of the attacking water molecule, and the PO l bond of the substrate (O a and O l stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of O a H and PO l bonds occurs simultaneously, whereas in the sequential mechanism, the O a H and PO l bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and deprotonated cases, the breaking of the water O a H bond occurs before the breaking of the PO l bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as semi-concurrent. The overall energy barrier is 41kcalmol -1 in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water O a H bond. This component is lowered from 34 to 25kcalmol -1 by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the PO l bond and is not sensitive to protonation. This is consistent with previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water O a H bond and to have little effect on the PO l bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the PO l bond could not be found here. This is because the leaving group in phosphate monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the �

Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

Science.gov (United States)

Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

2014-07-15

A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

Adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided versus empirical chemotherapy in unresectable non-small cell lung cancer.

Science.gov (United States)

Moon, Yong Wha; Sohn, Joo Hyuk; Kim, Yong Tai; Chang, Hyun; Jeong, Jae Heon; Lee, Young Joo; Chang, Joon; Kim, Se Kyu; Jung, Minkyu; Hong, Soojung; Choi, Sung Ho; Kim, Joo-Hang

2009-10-01

We retrospectively compared adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided and empirical chemotherapies for unresectable non-small cell lung cancer (NSCLC) in this case-control study. Unresectable NSCLC patients receiving ATP-CRA-guided platinum-based doublets as first-line therapy were enrolled as cases (n=27; 14 platinum-sensitive and 13 platinum-resistant patients). Performance status, stage, and chemotherapeutic regimen-matched patients receiving empirical chemotherapy were selected from the retrospective database as controls (n=93) in a case to control ratio of approximately 1:3. Response rate and survival (progression-free; overall) in both groups were not significantly different. However, the platinum-sensitive subgroup by ATP-CRA showed a higher response rate than the empirical group (71 versus 38%; p=0.023) with a trend toward longer progression-free survival (8.7 versus 4.8 months for platinum-sensitive versus empirical; p=0.223) and overall survival (not reached versus 12.6 months for platinum-sensitive versus empirical for p=0.134). ATP-CRA may be helpful in selecting platinum-responsive patients in unresectable NSCLC. We consider that nonplatinum doublets in platinum-resistant patients by ATP-CRA may be a more adapted approach than platinum-based doublets in future clinical trials.

A feasibility study of adenosine triphosphate-based chemotherapy response assay (ATP-CRA) as a chemosensitivity test for lung cancer.

Science.gov (United States)

Kang, Shin Myung; Park, Moo Suk; Chang, Joon; Kim, Se Kyu; Kim, Haeryoung; Shin, Dong-Hwan; Chung, Kyung Young; Kim, Dae Joon; Sohn, Joo Hyuk; Choi, Sung Ho; Kim, Jeongmi; Yoon, Eun Jin; Kim, Joo-Hang

2005-08-01

A chemosensitivity test can reflect the differences in responses of individual cancer patients to chemotherapeutic agents. The adenosine triphosphate-based chemotherapy response assay (ATP-CRA) is an accurate method, which does not require a large amount of tissue specimen. So far, no studies have evaluated the utility of the ATP-CRA in Korea. Therefore, we investigated the clinical usefulness of the ATP-CRA in 53 patients with lung cancer. Tumor tissues were obtained from bronchoscopic biopsies or surgical resections. The validity of ATP-CRA was assessed focusing on the success rate, experimental error level (intraassay mean coefficient of variation [CV]) and reproducibility. The overall success rate of ATP-CRA was 90.6% (48/53). Normal cells were effectively eliminated from the tumor tissues with the use of ficoll gradient centrifugation and immunomagnetic separation, which was confirmed using loss of heterozygosity analysis of the 3p deletion. The mean CV of ATP assays was 10.5+/-4.6%. The reproducibility of ATP assays was 94+/-3.8%. The results of the ATP assays were reported to physicians within 7 days of specimen collection. More than 6 anticancer drugs were tested on the tumor specimens obtained from bronchoscopic biopsies. The ATP-CRA is a stable, accurate and potentially practical chemosensitivity test in patients with lung cancer.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

Science.gov (United States)

Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

1995-08-04

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation.

Science.gov (United States)

Faustin, Benjamin; Lartigue, Lydia; Bruey, Jean-Marie; Luciano, Frederic; Sergienko, Eduard; Bailly-Maitre, Beatrice; Volkmann, Niels; Hanein, Dorit; Rouiller, Isabelle; Reed, John C

2007-03-09

Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.

Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

Science.gov (United States)

Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

2015-01-01

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

Activity of SHIP, Which Prevents Expression of Interleukin 1β, Is Reduced in Patients With Crohn's Disease.

Science.gov (United States)

Ngoh, Eyler N; Weisser, Shelley B; Lo, Young; Kozicky, Lisa K; Jen, Roger; Brugger, Hayley K; Menzies, Susan C; McLarren, Keith W; Nackiewicz, Dominika; van Rooijen, Nico; Jacobson, Kevan; Ehses, Jan A; Turvey, Stuart E; Sly, Laura M

2016-02-01

Crohn's disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5'-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d(-/-) mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. We collected intestines from SHIP-null mice, as well as Inpp5d(+/+) mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1β, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1β production was measured, and mechanisms of increased IL1β production were investigated. Macrophages were incubated with pan-phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1β production were measured. Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

Science.gov (United States)

Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

2014-11-01

The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

Artificial oxygen carrier with pharmacologic actions of adenosine-5'-triphosphate, adenosine, and reduced glutathione formulated to treat an array of medical conditions.

Science.gov (United States)

Simoni, Jan; Simoni, Grace; Moeller, John F; Feola, Mario; Wesson, Donald E

2014-08-01

Effective artificial oxygen carriers may offer a solution to tackling current transfusion medicine challenges such as blood shortages, red blood cell storage lesions, and transmission of emerging pathogens. These products, could provide additional therapeutic benefits besides oxygen delivery for an array of medical conditions. To meet these needs, we developed a hemoglobin (Hb)-based oxygen carrier, HemoTech, which utilizes the concept of pharmacologic cross-linking. It consists of purified bovine Hb cross-linked intramolecularly with open ring adenosine-5'-triphosphate (ATP) and intermolecularly with open ring adenosine, and conjugated with reduced glutathione (GSH). In this composition, ATP prevents Hb dimerization, and adenosine promotes formation of Hb polymers as well as counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine receptors. ATP also serves as a regulator of vascular tone through activation of purinergic receptors. GSH blocks Hb's extravasation and glomerular filtration by lowering the isoelectric point, as well as shields heme from nitric oxide and reactive oxygen species. HemoTech and its manufacturing technology have been broadly tested, including viral and prion clearance validation studies and various nonclinical pharmacology, toxicology, genotoxicity, and efficacy tests. The clinical proof-of-concept was carried out in sickle cell anemia subjects. The preclinical and clinical studies indicate that HemoTech works as a physiologic oxygen carrier and has efficacy in treating: (i) acute blood loss anemia by providing a temporary oxygen bridge while stimulating an endogenous erythropoietic response; (ii) sickle cell disease by counteracting vaso-occlusive/inflammatory episodes and anemia; and (iii) ischemic vascular diseases particularly thrombotic and restenotic events. The pharmacologic cross-linking of Hb with ATP, adenosine, and GSH showed usefulness in designing an artificial oxygen carrier for

Role of hemolysis in red cell adenosine triphosphate release in simulated exercise conditions in vitro.

Science.gov (United States)

Mairbäurl, Heimo; Ruppe, Florian A; Bärtsch, Peter

2013-10-01

Specific adenosine triphosphate (ATP) release from red blood cells has been discussed as a possible mediator controlling microcirculation in states of decreased tissue oxygen. Because intravascular hemolysis might also contribute to plasma ATP, we tested in vitro which portion of ATP release is due to hemolysis in typical exercise-induced strains to the red blood cells (shear stress, deoxygenation, and lactic acidosis). Human erythrocytes were suspended in dextran-containing media (hematocrit 10%) and were exposed to shear stress in a rotating Couette viscometer at 37°C. Desaturation (oxygen saturation of hemoglobin ∼20%) was achieved by tonometry with N2 before shear stress exposure. Cells not exposed to shear stress were used as controls. Na lactate (15 mM), lactic acid (15 mM, pH 7.0), and HCl (pH 7.0) were added to simulate exercise-induced lactic acidosis. After incubation, extracellular hemoglobin was measured to quantify hemolysis. ATP was measured with the luciferase assay. Shear stress increased extracellular ATP in a stress-related and time-dependent manner. Hypoxia induced a ∼10-fold increase in extracellular ATP in nonsheared cells and shear stress-exposed cells. Lactic acid had no significant effect on ATP release and hemolysis. In normoxic cells, approximately 20%-50% of extracellular ATP was due to hemolysis. This proportion decreased to less than 10% in hypoxic cells. Our results indicate that when exposing red blood cells to typical strains they encounter when passing through capillaries of exercising skeletal muscle, ATP release from red blood cells is caused mainly by deoxygenation and shear stress, whereas lactic acidosis had only a minor effect. Hemolysis effects were decreased when hemoglobin was deoxygenated. Together, by specific release and hemolysis, extracellular ATP reaches values that have been shown to cause local vasodilatation.

Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage.

Science.gov (United States)

de Korte, Dirk; Kleine, Mya; Korsten, Herbert G H; Verhoeven, Arthur J

2008-06-01

Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.

Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

International Nuclear Information System (INIS)

Hunting, D.J.; Dresler, S.L.

1985-01-01

We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord [v1; ref status: indexed, http://f1000r.es/3rm

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Eric McCoy

2014-07-01

Full Text Available Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides.  Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP to adenosine in primary somatosensory neurons.  Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons.  Ectonucleoside triphosphate diphosphohydrolases (ENTPDs comprise a class of enzymes that dephosphorylate extracellular ATP and ADP.  Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3 was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG, in the dorsal horn of the spinal cord, and in free nerve endings in the skin.  To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse.  This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder.  However, DRG and spinal cord tissues from Entpd3-/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates.  Additionally, using fast-scan cyclic voltammetry (FSCV, adenosine production was not impaired in the dorsal spinal cord of Entpd3-/- mice when the substrate ADP was applied.  Further, Entpd3-/- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3-/- mice showed a modest reduction in β-alanine-mediated itch.  Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord.  Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.

Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord [v2; ref status: indexed, http://f1000r.es/4dl

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Eric McCoy

2014-09-01

Full Text Available Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides.  Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP to adenosine in primary somatosensory neurons.  Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons.  Ectonucleoside triphosphate diphosphohydrolases (ENTPDs comprise a class of enzymes that dephosphorylate extracellular ATP and ADP.  Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3 was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG, in the dorsal horn of the spinal cord, and in free nerve endings in the skin.  To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse.  This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder.  However, DRG and spinal cord tissues from Entpd3-/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates.  Additionally, using fast-scan cyclic voltammetry (FSCV, adenosine production was not impaired in the dorsal spinal cord of Entpd3-/- mice when the substrate ADP was applied.  Further, Entpd3-/- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3-/- mice showed a modest reduction in β-alanine-mediated itch.  Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord.  Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.

Depression of presynaptic excitation by the activation of vanilloid receptor 1 in the rat spinal dorsal horn revealed by optical imaging

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Ikeda Hiroshi

2006-02-01

Full Text Available Abstract In this study, we show that capsaicin (CAP depresses primary afferent fiber terminal excitability by acting on vanilloid receptor 1 (TRPV1 channels of primary afferent fibers in adenosine 5'-triphosphate (ATP- and temperature-dependent manner using two optical imaging methods. First, transverse slices of spinal cord were stained with a voltage-sensitive dye and the net excitation in the spinal dorsal horn was recorded. Prolonged treatment (>20 min with the TRPV1 channel agonist, CAP, resulted in a long-lasting inhibition of the net excitation evoked by single-pulse stimulation of C fiber-activating strength. A shorter application of CAP inhibited the excitation in a concentration-dependent manner and the inhibition was reversed within several minutes. This inhibition was Ca++-dependent, was antagonized by the TRPV1 channel antagonist, capsazepine (CPZ, and the P2X and P2Y antagonist, suramin, and was facilitated by the P2Y agonist, uridine 5'-triphosphate (UTP. The inhibition of excitation was unaffected by bicuculline and strychnine, antagonists of GABAA and glycine receptors, respectively. Raising the perfusate temperature to 39°C from 27°C inhibited the excitation (-3%/°C. This depressant effect was antagonized by CPZ and suramin, but not by the P2X antagonist, 2', 3'-O-(2,4,6-trinitrophenyl adenosine 5'-triphosphate (TNP-ATP. Second, in order to record the presynaptic excitation exclusively, we stained the primary afferent fibers anterogradely from the dorsal root. CAP application and a temperature increase from 27°C to 33°C depressed the presynaptic excitation, and CPZ antagonized these effects. Thus, this study showed that presynaptic excitability is modulated by CAP, temperature, and ATP under physiological conditions, and explains the reported central actions of CAP. These results may have clinical importance, especially for the control of pain.

Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of Culicoides variipennis, the North American vector of bluetongue viruses.

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Pérez de León, A A; Tabachnick, W J

1996-02-01

Salivary gland homogenates of Culicoides variipennis, the primary vector of bluetongue (BLU) viruses in North America, were analyzed for apyrase activity. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is an anti-hemostatic and anti-inflammatory salivary enzyme of most hematophagous arthropods. The enzyme activity was measured by the release of orthophosphate using ATP, ADP, and AMP as substrates with Ca2+ as the divalent cation. ATPase (11.5 +/- 1 mU/pair of glands), ADPase (7.3 +/- 0.7 mU/pair of glands), and insignificant (P < 0.05) AMPase (0.07 mU/pair of glands) activities were detected in female salivary glands. Male salivary glands contained lower amounts of ATPase and ADPase activity (P < 0.05). The ATPase and ADPase activities were greatest at pH 8.5, and were similarly activated by Mg2+. Molecular sieving HPLC of salivary gland homogenates generated a single peak which coincided with ATPase and ADPase, but no AMPase, activity; the protein has an estimated molecular mass of 35,000 Da. ATPase and ADPase activity, and total protein concentration, were reduced (P < 0.05) in the salivary glands of females after taking a blood meal from a sheep. Salivary gland homogenates also inhibited ADP-induced platelet aggregation in vitro. It is concluded that the salivary ATPase and ADPase activities of C. variipennis reside in one enzyme, and that this enzyme is likely an apyrase. The apyrase activity is thought to be responsible for the inhibition of ADP-induced platelet aggregation, as indicated by the apparent discharge of apyrase from salivary glands into the host during blood feeding. This suggests that apyrase is one of the salivary proteins present in C. variipennis acting as antigens in the development of Culicoides hypersensitivity in ruminants and horses. Apyrase may inhibit an inflammatory response at the feeding site through the subsequent degradation of its end-product, AMP, to adenosine, a potent anti-inflammatory substance, by the ecto-5' nucleotidase

Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

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Benediktsson, I.; Spampinato, C.P.; Andreo, C.S.; Schieder, O.

1994-01-01

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the role of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity. (author)

5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

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Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

2004-01-01

The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

Mechanism of action of minoxidil in the treatment of androgenetic alopecia is likely mediated by mitochondrial adenosine triphosphate synthase-induced stem cell differentiation.

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Goren, A; Naccarato, T; Situm, M; Kovacevic, M; Lotti, T; McCoy, J

2017-01-01

Topical minoxidil is the only topical drug approved by the US Food and Drug Administration (FDA) for the treatment of androgenetic alopecia. However, the exact mechanism by which minoxidil stimulates anagen phase and promotes hair growth is not fully understood. In the late telegen phase of the hair follicle growth cycle, stem cells located in the bulge region differentiate and re-enter anagen phase, a period of growth lasting 2-6 years. In androgenetic alopecia, the anagen phase is shortened and a progressive miniaturization of hair follicles occurs, eventually leading to hair loss. Several studies have demonstrated that minoxidil increases the amount of intracellular Ca2+, which has been shown to up-regulate the enzyme adenosine triphosphate (ATP) synthase. A recent study demonstrated that ATP synthase, independent of its role in ATP synthesis, promotes stem cell differentiation. As such, we propose that minoxidil induced Ca2+ influx can increase stem cell differentiation and may be a key factor in the mechanism by which minoxidil facilitates hair growth. Based on our theory, we provide a roadmap for the development of a new class of drugs for the treatment of androgenetic alopecia.

Juvenile Hormone Analogues, Methoprene and Fenoxycarb Dose-Dependently Enhance Certain Enzyme Activities in the Silkworm Bombyx Mori (L

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M. Rajeswara Rao

2008-06-01

Full Text Available Use of Juvenile Hormone Analogues (JHA in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 μg and 3.0fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT and alanine aminotransaminase (ALAT, adenosine triphosphate synthase (ATPase and cytochrome-c-oxidase (CCO activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues.

Down-regulation of NTPDase2 and ADP-sensitive P2 Purinoceptors Correlate with Severity of Symptoms during Experimental Autoimmune Encephalomyelitis

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Marija Jakovljevic

2017-10-01

Full Text Available The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2 and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE. Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset (Eo, peak (Ep and recovery (Er from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP-immunoreactive (ir astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er. The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5′-nucleotidase (eN/CD73 was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4− cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which

Expression of adenosine triphosphate-sensitive potassium channels in rats with cirrhosis: correlationship with sympathetic activity and renal function

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Julio Cesar Martins Monte

2006-12-01

Full Text Available Objective: The aim of this study was to perform a direct analysis ofKATP mRNA expression by RT-PCR in kidney and isolated aorta fromrats with cirrhosis (induced by carbon tetrachloride and controls.The present study also analyses the relation between induced cirrhosisand urinary excretion of sodium and sympathetic activity in cirrhoticrats. Methods: Rats were placed in metabolic cages and allowedfree access to food and water. Cirrhosis was induced by repeateddoses of carbon tetrachloride by gastric gavage. After some weeks,the kidney and aorta were dissected and utilized for RNA extraction.Blood and urine were analyzed for electrolytes. Renal function wasestimated by creatinine clearance and sodium urinary excretion.Serum catecholamines were measured by HPLC analysis. Results:First, RT-PCR analysis showed that KATP mRNA is expressed in liverwith cirrhosis and intense fibrosis, but not with moderate fibrosis.Second, RT-PCR analysis revealed that KATP mRNA was detectedonly in aorta dissected from rats with cirrhosis. Finally, an enhancedreabsorption of sodium without renal failure suggests a potentialmediator would increase the activity of the sympathetic system.Conclusion: These results suggest that KATP mRNA is expressed incirrhotic rats with sympathetic activation and renal dysfunction. Thischannel might be involved in another route where the vascular tonecan be modulated in cirrhosis.

[A prospective study of adenosine triphosphate-tumor chemosensitivity assay directed chemotherapy in patients with recurrent ovarian cancer].

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Gao, Yu-tao; Wu, Ling-ying; Zhang, Wei; Zhao, Dan; Li, Ning; Tian, Hai-mei; Wang, Xiao-bing; Li, Mo; Sun, Yang-chun; Li, Nan; Li, Xiao-guang

2013-05-01

To investigate the efficacy of adenosine triphosphate (ATP)-tumor chemosensitivity assay (TCA) directed chemotherapy in patients with recurrent epithelial ovarian cancer. From August 2010 to June 2012, recurrent epithelial ovarian cancer patients were prospectively enrollmented in Cancer Hospital, Peking Union Medical College,Chinese Academy of Medical Sciences.The entry criteria are as follows: (1) Histologically proven to be epithelial ovarian cancer. (2) Patients of recurrent ovarian cancer with bidimensionally measurable tumor, or ascitic or pleural fluid for testing. (3) Karnofsky performance status > 60. (4) A life expectancy of at least more than 6 months.According to patients desires, they were assigned into two groups: assay-directed therapy group and physician's-choice therapy group, patients' clinical and pathological characteristics, response rate to chemotherapy and progression-free survival (PFS) were compared between two groups. A total of 113 patients with recurrent epithelial ovarian cancer were prospectively enrollmented to assay-directed chemotherapy (n = 56) or physician's-choice chemotherapy (n = 57).There was no difference in median age,types of recurrence, surgical-pathological stage, pathological type, tumor grade, times of recurrence, residual disease at secondary cytoreductive surgery between assay-directed group and physician's-choice group. The overall response rate (ORR) and median PFS in the ATP-TCA group was 66% (37/56) and 7 months, while the ORR in the control group was 46% (26/57, P = 0.037), the median PFS was 4 months (P = 0.040). For platinum-resistant patients, the ORR between ATP-TCA directed chemotherapy 59% (16/27) and control group 25% (7/28) were significantly different (P = 0.010), and the median PFS between two groups were also significantly different (5 months and 2 months, respectively, P = 0.003). ATP-TCA directed chemotherapy could improve ORR and PFS in patients with recurrent epithelial ovarian cancer, especially

Comparison of myocardial blood flow induced by adenosine triphosphate and dipyridamole in patients with coronary artery disease

International Nuclear Information System (INIS)

Mamede, M.; Tadamura, Eiji; Hosokawa, Ryohei

2005-01-01

Myocardial perfusion imaging with adenosine triphosphate (ATP) has been used increasingly to diagnose coronary artery disease (CAD) and assess risk for this disease. This study compared absolute myocardial blood flow (MBF) and myocardial flow reserve index (MFR) with ATP and dipyridamole (DIP) in patients with CAD. MBF was quantified by 15 O-H 2 O PET in 21 patients with CAD (17 male, 4 female), aged 55 to 81 years. MBF was measured at rest, during intravenous injection of ATP (0.16 mg/kg/min), and again after DIP infusion (0.56 mg/kg). Regions of interest were drawn in nonischemic and ischemic segments based on findings from thallium-201 ( 201 Tl) scintigraphy and coronary angiography (CAG). Absolute MBF values and indexes of MFR were calculated in nonischemic and ischemic segments. Intravenous injection of ATP and DIP significantly increased MBF in nonischemic (2.4±0.9 and 2.1±0.8 ml/g/min, respectively; p<0.01, for both) and in ischemic segments (1.3±0.4 and 1.5±0.4 ml/g/min, respectively; p<0.01, for both). There was a significant difference in MBF values between ATP and DIP in nonischemic segments (p<0.05), which was not observed in ischemic segments. In nonischemic segments, ATP produced higher MFR than DIP (2.1±0.8 and 1.8±0.7, respectively; p<0.05), while no significant difference was observed in ischemic segments (1.5±0.6 and 1.7±0.3, respectively). ATP produced a greater hyperemia than DIP between the ischemic and nonischemic myocardium in patients with CAD. ATP is as effective as DIP for the diagnosis of CAD. (author)

T cell activity in successful treatment of chronic urticaria with omalizumab

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Gonzalez Ruperto

2011-07-01

Full Text Available Abstract Omalizumab, a humanized monoclonal anti-IgE antibody has the potential to alter allergen processing. Recently, it has been postulated the assessment of PHA-stimulated adenosine triphosphate (ATP activity as maker of CD4+ T cells activity in peripheral blood cells. We present the case report of a 35-year-old woman with a history of chronic idiopathic urticaria and angioedema of 8 years of development with poor response to treatment. The patient was partially controlled with cyclosporine at doses of 100 mg/12 h. However, she was still developing hives daily. Finally treatment with omalizumab was started at dose of 300 mg every 2 weeks. The patient experienced a decrease in urticarial lesions 2 days after starting therapy. We also evaluated the effects of omalizumab therapy on the activity of peripheral blood CD4+ T cells from the patient, in order to determine the potential modification of anti-IgE therapy on the process of antigen presentation-recognition. Activity of CD4+ cells by ATP release was clearly increased demonstrating an enlarged CD4 activity. Omalizumab may be useful in the treatment of severe chronic urticaria. ATP activity of peripheral blood CD4+ T cells might be a non-subjective method to assess Omalizumab activity.

An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

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Rubinstein, D; Warrendorf, E

1975-06-01

The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.

Yolk-Shell Porous Microspheres of Calcium Phosphate Prepared by Using Calcium L-Lactate and Adenosine 5'-Triphosphate Disodium Salt: Application in Protein/Drug Delivery.

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Ding, Guan-Jun; Zhu, Ying-Jie; Qi, Chao; Sun, Tuan-Wei; Wu, Jin; Chen, Feng

2015-06-26

A facile and environmentally friendly approach has been developed to prepare yolk-shell porous microspheres of calcium phosphate by using calcium L-lactate pentahydrate (CL) as the calcium source and adenosine 5'-triphosphate disodium salt (ATP) as the phosphate source through the microwave-assisted hydrothermal method. The effects of the concentration of CL, the microwave hydrothermal temperature, and the time on the morphology and crystal phase of the product are investigated. The possible formation mechanism of yolk-shell porous microspheres of calcium phosphate is proposed. Hemoglobin from bovine red cells (Hb) and ibuprofen (IBU) are used to explore the application potential of yolk-shell porous microspheres of calcium phosphate in protein/drug loading and delivery. The experimental results indicate that the as-prepared yolk-shell porous microspheres of calcium phosphate have relatively high protein/drug loading capacity, sustained protein/drug release, favorable pH-responsive release behavior, and a high biocompatibility in the cytotoxicity test. Therefore, the yolk-shell porous microspheres of calcium phosphate have promising applications in various biomedical fields such as protein/drug delivery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

Science.gov (United States)

Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

2013-10-01

Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

Spectral studies of lanthanide-nucleic acid component interaction: complexes of adenine, adenosine, adenosine 5'-mono-, adenosine 5'-di- and adenosine 5' tri-phosphates with praseodymium(III)

International Nuclear Information System (INIS)

Joseph, George; Anjaiah, K.; Misra, S.N.

1990-01-01

The interactions of adenine, adenosine, adenosine 5'-mono-, adenosine 5'-di-and adenosine 5'-tri-phosphates with praseodymium(III) have been studied in different stoichiometries and at varying hydrogen ion concentrations by absorption spectral studies. The sharp bands in the spectra have been individually analysed by Gaussian curve analysis, and various spectral parameters have been computed using partial and multiple regression methods on an HP-1000/45 computer. The changes in and the magnitudes of these parameters have been correlated with the degrees of outer- and inner-sphere coordination around praseodymium(III). Crystalline complexes of the type: Pr(nucleotide) 2 (H 2 O) 2 (where nucleotide = AMP, ADP and ATP) have been characterized on the basis of analytical, IR and 1 H NMR spectral data. These studies indicate that the binding of the nucleotide is through phosphoric oxygen. These complexes in aqueous medium show significant ionization which supports the observed weak 4f-4f bands, lower values of nephelauxetic effect and the parameters derived from coulombic and spin-orbit interactions. (author). 3 t abs., 28 refs

Herpes simplex virus triggers activation of calcium-signaling pathways

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Cheshenko, Natalia; Del Rosario, Brian; Woda, Craig; Marcellino, Daniel; Satlin, Lisa M.; Herold, Betsy C.

2003-01-01

The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)–sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy. PMID:14568989

Safety and feasibility of thallium-201 myocardial SPECT with intravenous infusion of disodium adenosine triphosphate (ATP) in the diagnosis of coronary artery disease

Energy Technology Data Exchange (ETDEWEB)

Pai, Moon Sun; Park, Chan H.; Yoon, Seok Nam; Kim, Won; Kim, Han Soo [College of Medicine, Ajou Univ., Suwon (Korea, Republic of)

1998-08-01

ATP (adenosine triphosphate) is a potent coronary vasodilator with a rapid onset of action and a very short half-life. Myocardial perfusion scintigraphy with intravenous ATP has not yet bee sufficiently proven in the diagnosis, follow-up, and risk stratification of coronary artery disease. The purpose of this study was to evaluate the safety, feasibility and diagnostic accuracy of pharmacologic stress thallium-102 myocardial SPECT using an intravenous ATP infusion in patients with suspected coronary artery disease. Thallium-201 myocardial SPECT in 319 patients with suspected coronary artery disease were performed after the infusion of ATP (0.08 mg/min for 6 min). The adverse effects were carefully monitored. Coronary angiography was also performed within 3 weeks. Although 76.5% of he patients had some adverse effects, they were transient, mild, and well tolerated. In all patients, the ATP infusion protocol was completed and only 2 patients required aminophylline. The adverse effects were dyspnea in 63%, headache in 31%, flushing in 21%, chest pain in 14% and abdominal discomfort in 5% of the patients. The sensitivity and specificity were 80% and 90% respectively. Thallium-201 myocardial SPECT after 6 min-infusion of ATP at a rate of 0.08 mg/kg/min is safe and has a diagnostic value in detecting coronary artery disease.

Safety and feasibility of thallium-201 myocardial SPECT with intravenous infusion of disodium adenosine triphosphate (ATP) in the diagnosis of coronary artery disease

International Nuclear Information System (INIS)

Pai, Moon Sun; Park, Chan H.; Yoon, Seok Nam; Kim, Won; Kim, Han Soo

1998-01-01

ATP (adenosine triphosphate) is a potent coronary vasodilator with a rapid onset of action and a very short half-life. Myocardial perfusion scintigraphy with intravenous ATP has not yet bee sufficiently proven in the diagnosis, follow-up, and risk stratification of coronary artery disease. The purpose of this study was to evaluate the safety, feasibility and diagnostic accuracy of pharmacologic stress thallium-102 myocardial SPECT using an intravenous ATP infusion in patients with suspected coronary artery disease. Thallium-201 myocardial SPECT in 319 patients with suspected coronary artery disease were performed after the infusion of ATP (0.08 mg/min for 6 min). The adverse effects were carefully monitored. Coronary angiography was also performed within 3 weeks. Although 76.5% of he patients had some adverse effects, they were transient, mild, and well tolerated. In all patients, the ATP infusion protocol was completed and only 2 patients required aminophylline. The adverse effects were dyspnea in 63%, headache in 31%, flushing in 21%, chest pain in 14% and abdominal discomfort in 5% of the patients. The sensitivity and specificity were 80% and 90% respectively. Thallium-201 myocardial SPECT after 6 min-infusion of ATP at a rate of 0.08 mg/kg/min is safe and has a diagnostic value in detecting coronary artery disease

Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

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Myrna eBarjau Perez-Milicua

2015-07-01

Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing.

Science.gov (United States)

Liang, Ru-Ping; Qiu, Wei-Bin; Zhao, Hui-Fang; Xiang, Cai-Yun; Qiu, Jian-Ding

2016-01-21

Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody-antigen interaction in the presence of casein kinase II (CK2) and adenosine 5'-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Monitoring of intracellular adenosine triphosphate in CD4(+) T cells to predict the occurrence of cytomegalovirus disease in kidney transplant recipients.

Science.gov (United States)

Pérez-Jacoiste Asín, María Asunción; Fernández-Ruiz, Mario; López-Medrano, Francisco; Aquilino, Carolina; González, Esther; Ruiz-Merlo, Tamara; Gutiérrez, Eduardo; San Juan, Rafael; Paz-Artal, Estela; Andrés, Amado; Aguado, José Maria

2016-10-01

The measurement of intracellular concentrations of adenosine triphosphate (iATP) in phytohemagglutinin-stimulated CD4(+) T cells constitutes a surrogate marker for post-transplant cell-mediated immunity (CMI). This assay has shown suboptimal accuracy for predicting infection after kidney transplantation (KT). We hypothesize that its predictive capacity depends on the specific contribution of the CMI to host-pathogen interactions. We assessed iATP levels in 100 KT recipients at baseline and months 1, 3, and 6 (363 measurements). No association was found between iATP at month 1 and the risk for overall or bacterial infection, although such association was evident for cytomegalovirus (CMV) disease (multivariate-adjusted hazard ratio [per 50-unit increment]: 0.83; P-value = 0.048). There were no significant differences in mean iATP between stable patients (319.4 ng/ml) and those developing overall (304.1 ng/ml) or bacterial infection (346.9 ng/ml) over the 45 days following monitoring. However, iATP was significantly lower in patients who developed CMV disease (223.5 ng/ml; P-values <0.002). The optimal cutoff (265 ng/ml) for predicting CMV disease in patients not receiving antiviral prophylaxis yielded sensitivity, specificity, positive, and negative predictive values of 85.7%, 68.3%, 15.2%, and 98.6%, respectively. In conclusion, a non-pathogen-specific monitoring of CMI by means of iATP informs the risk of CMV disease in KT recipients. © 2016 Steunstichting ESOT.

Coamplification in tumors of KRAS2, type 2 inositol 1,4,5 triphosphate receptor gene, and a novel human gene, KRAG

Energy Technology Data Exchange (ETDEWEB)

Heighway, J.; Betticher, D.C.; Altermatt, H.J. [Univ. Hospital of Berne (Switzerland)] [and others

1996-07-01

Analysis of a region of DNA, coamplified in tumors with KRAS2, resulted in the identification of the human homologue of the mouse KRAG gene. The gene was widely expressed in range of cell lines, tumors, and normal tissue and demonstrated a high degree of alternate splicing. A human KRAG cDNA sequence, with a structure similar to that encoded by the amplified gene in mouse Y1 adrenal carcinoma cells, was isolated by RT-PCR. The predicted amino acid similarity between the two sequences was 91%, and hydrophobicity plots suggested a structure closely resembling that of transmembrane 4 superfamily members. Identification of a PCR-based restriction fragment length polymorphism allele-specific splicing differences in tumors. Northern analysis of mRNA derived from a range of tissues suggested high level expression in muscle and confirmed alternate splicing. To facilitate the analysis of exon junctions, a YAC clone encoding the genomic sequence was identified. This allowed the localization of KRAG to human chromosome 12p11.2. Isolation of one end of this nonchimeric clone demonstrated a perfect match with a 247-bp sequence within the 3{prime} untranslated region of the type 2 1,4,5-inositol triphosphate receptor gene. Multiplex PCR confirmed the inclusion of both genes. Multiplex PCR confirmed the inclusion of both genes in the KRAS2 amplicon in human malignancy, suggesting that either may contribute to the malignant phenotypes. 35 refs., 6 figs., 1 tab.

Bacterial diversity and active biomass in full-scale granular activated carbon filters operated at low water temperatures.

Science.gov (United States)

Kaarela, Outi E; Härkki, Heli A; Palmroth, Marja R T; Tuhkanen, Tuula A

2015-01-01

Granular activated carbon (GAC) filtration enhances the removal of natural organic matter and micropollutants in drinking water treatment. Microbial communities in GAC filters contribute to the removal of the biodegradable part of organic matter, and thus help to control microbial regrowth in the distribution system. Our objectives were to investigate bacterial community dynamics, identify the major bacterial groups, and determine the concentration of active bacterial biomass in full-scale GAC filters treating cold (3.7-9.5°C), physicochemically pretreated, and ozonated lake water. Three sampling rounds were conducted to study six GAC filters of different operation times and flow modes in winter, spring, and summer. Total organic carbon results indicated that both the first-step and second-step filters contributed to the removal of organic matter. Length heterogeneity analysis of amplified 16S rRNA genes illustrated that bacterial communities were diverse and considerably stable over time. α-Proteobacteria, β-Proteobacteria, and Nitrospira dominated in all of the GAC filters, although the relative proportion of dominant phylogenetic groups in individual filters differed. The active bacterial biomass accumulation, measured as adenosine triphosphate, was limited due to low temperature, low flux of nutrients, and frequent backwashing. The concentration of active bacterial biomass was not affected by the moderate seasonal temperature variation. In summary, the results provided an insight into the biological component of GAC filtration in cold water temperatures and the operational parameters affecting it.

Visual and surface plasmon resonance sensor for zirconium based on zirconium-induced aggregation of adenosine triphosphate-stabilized gold nanoparticles

International Nuclear Information System (INIS)

Qi, Wenjing; Zhao, Jianming; Zhang, Wei; Liu, Zhongyuan; Xu, Min; Anjum, Saima; Majeed, Saadat; Xu, Guobao

2013-01-01

Graphical abstract: Visual and surface plasmon resonance (SPR) sensor for Zr(IV) has been developed for the first time based on Zr(IV)-induced change of SPR absorption spectra of ATP-stabilized AuNP solutions. -- Highlights: •Visual and SPR absorption Zr 4+ sensors have been developed for the first time. •The high affinity between Zr 4+ and ATP makes sensor highly sensitive and selective. •A fast response to Zr 4+ within 4 min. -- Abstract: Owing to its high affinity with phosphate, Zr(IV) can induce the aggregation of adenosine 5′-triphosphate (ATP)-stabilized AuNPs, leading to the change of surface plasmon resonance (SPR) absorption spectra and color of ATP-stabilized AuNP solutions. Based on these phenomena, visual and SPR sensors for Zr(IV) have been developed for the first time. The A 660 nm /A 518 nm values of ATP-stabilized AuNPs in SPR absorption spectra increase linearly with the concentrations of Zr(IV) from 0.5 μM to 100 μM (r = 0.9971) with a detection limit of 95 nM. A visual Zr(IV) detection is achieved with a detection limit of 30 μM. The sensor shows excellent selectivity against other metal ions, such as Cu 2+ , Fe 3+ , Cd 2+ , and Pb 2+ . The recoveries for the detection of 5 μM, 10 μM, 25 μM and 75 μM Zr(IV) in lake water samples are 96.0%, 97.0%, 95.6% and 102.4%, respectively. The recoveries of the proposed SPR method are comparable with those of ICP-OES method

A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

Science.gov (United States)

Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

2014-08-07

A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone.

Science.gov (United States)

Detremmerie, Charlotte; Vanhoutte, Paul M; Leung, Susan

2017-07-01

The natural compound thymoquinone, extracted from Nigella sativa (black cumin), is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC) producing inosine 3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine 3',5'-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

Adenyl cyclase activator forskolin protects against Huntington's disease-like neurodegenerative disorders

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Sidharth Mehan

2017-01-01

Full Text Available Long term suppression of succinate dehydrogenase by selective inhibitor 3-nitropropionic acid has been used in rodents to model Huntington's disease where mitochondrial dysfunction and oxidative damages are primary pathological hallmarks for neuronal damage. Improvements in learning and memory abilities, recovery of energy levels, and reduction of excitotoxicity damage can be achieved through activation of Adenyl cyclase enzyme by a specific phytochemical forskolin. In this study, intraperitoneal administration of 10 mg/kg 3-nitropropionic acid for 15 days in rats notably reduced body weight, worsened motor cocordination (grip strength, beam crossing task, locomotor activity, resulted in learning and memory deficits, greatly increased acetylcholinesterase, lactate dehydrogenase, nitrite, and malondialdehyde levels, obviously decreased adenosine triphosphate, succinate dehydrogenase, superoxide dismutase, catalase, and reduced glutathione levels in the striatum, cortex and hippocampus. Intragastric administration of forskolin at 10, 20, 30 mg/kg dose-dependently reversed these behavioral, biochemical and pathological changes caused by 3-nitropropionic acid. These results suggest that forskolin exhibits neuroprotective effects on 3-nitropropionic acid-induced Huntington's disease-like neurodegeneration.

4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate (4PyTP, a Novel NAD+ Metabolite Accumulating in Erythrocytes of Uremic Children: A Biomarker for a Toxic NAD+ Analogue in Other Tissues?

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Elizabeth A. Carrey

2011-06-01

Full Text Available We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP, which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD+ metabolites (nicotinamide, N1-methylnicotinamide and 4Py-riboside and the major nicotinamide metabolite N1-methyl-2-pyridone-5-carboxamide (2PY, with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD+ from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD+ analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD+ analogue that inhibits IMP dehydrogenase in other cells.

Activity of acid phosphatase in tissues of dogs exposed to γ-radiation at low dose rates and treated with adenosine triphosphate

International Nuclear Information System (INIS)

Bichejkina, N.I.; Tikhomirova, M.V.; Romantsev, E.F.; Rogozkin, V.D.

1979-01-01

A study was made of the activity of acid phosphatase in the liver, spleen and blood of dogs under various experimental conditions: (a) exposure to γ-rays at low dose rates, (b) preventive and therapeutic application of ATP and (c) administration of ATP to intact animals. It was demonstrated that the activity of acid phosphatase in the liver and spleen was invariable after the first 24 h and decreased after 72 h of observation in each of the experimental variants. Preventive and therapeutic administration of ATP to dogs not substantially influence the activity of acid phosphatase throughout the entire period of observation

Anti-hypoxic activity of the ethanol extract from Portulaca oleracea in mice.

Science.gov (United States)

Chen, Cheng-Jie; Wang, Wan-Yin; Wang, Xiao-Li; Dong, Li-Wei; Yue, Yi-Tian; Xin, Hai-Liang; Ling, Chang-Quan; Li, Min

2009-07-15

To investigate the effects of the ethanol extract from Portulaca oleracea (EEPO) on hypoxia models mice and to find the possible mechanism of its anti-hypoxic actions so as to elucidate the anti-hypoxia activity and provide scientific basis for the clinical use of Portulaca oleracea. EEPO was evaluated on anti-hypoxic activity in several hypoxia mice models, including closed normobaric hypoxia and sodium nitrite or potassium cyanide toxicosis. To verify the possible mechanism(s), we detected the activities of pyruvate kinase (PK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and the level of adenosine triphosphate (ATP) in mice cortices. Given orally, the EEPO at doses of 100, 200, 400 mg/kg could dose-dependently enhance the survival time of mice in both of the normobaric and chemical hypoxia models. The activity of the glycolysis enzymes and the level of ATP were higher than those of the control. In the pentobarbital sodium-induced sleeping time test and the open-field test, EEPO neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-hypoxic activity was unlikely due to sedation or motor abnormality. These results demonstrated that the EEPO possessed notable anti-hypoxic activity, which might be related to promoting the activity of the key enzymes in glycolysis and improving the level of ATP in hypoxic mice.

Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

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Jerome Deval

2015-06-01

Full Text Available Respiratory syncytial virus (RSV causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV, whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

Science.gov (United States)

Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

2017-03-15

This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive determination of adenosine disodium triphosphate in soil, milk, and pharmaceutical formulation by enoxacin–europium (III) fluorescence complex in solution

International Nuclear Information System (INIS)

Alam, Al-Mahmnur; Kamruzzaman, Mohammad; Hak Lee, Sang; Ho Kim, Young; Jin Jo, Hae; Hong Kim, Sung; Park, Sang-Ryoul

2012-01-01

A new spectroflurometric method for the determination of adenosine disodium triphosphate (ATP) is developed. Fluorometric interaction between ATP and enoxacin (ENX)–Eu 3+ complex was studied using UV–vis and fluorescence spectroscopy. Weak luminescence spectra of Eu 3+ were enhanced after complexation with ENX at 589 nm and 614 nm upon excitation at 395 nm due to energy transfer from the ligand to the lanthanide ion. It was observed that luminescence spectrum of Eu 3+ was strongly enhanced further at 614 nm after incorporation of ATP into the ENX–Eu 3+ complex. Under optimal conditions, the enhancement of luminescence at 614 nm was responded linearly with the concentration of ATP. The linearity was maintained in the range of 1.5×10 −10 –1.15×10 −8 M (R=0.9973) with the limit of detection (3σ) of 4.71×10 −11 M. The relative standard deviation (RSD) for 9 repeated measurements of 1×10 −9 M ATP was 1.25%. Successful determinations of ATP in soil, milk, and a pharmaceutical formulation with the proposed method were demonstrated. - Highlights: ► Weak luminescence of Eu 3+ was enhanced at 614 nm after formation of complex with ENX. ► Energy transfer occurs through FRET from ENX to Eu 3+ upon excitation. ► Luminescence signal was further enhanced when ATP conjugates with ENX–Eu 3+ complex. ► Luminescence intensity of Eu 3+ at 614 nm was correlated with concentration of ATP. ► The method was applied to determine ATP in soil, milk, and pharmaceutical samples.

The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

Science.gov (United States)

McCully, Kilmer S

2015-01-01

The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. © 2015 by the Association of Clinical Scientists, Inc.

PDK1 Is a Regulator of Epidermal Differentiation that Activates and Organizes Asymmetric Cell Division

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Teruki Dainichi

2016-05-01

Full Text Available Asymmetric cell division (ACD in a perpendicular orientation promotes cell differentiation and organizes the stratified epithelium. However, the upstream cues regulating ACD have not been identified. Here, we report that phosphoinositide-dependent kinase 1 (PDK1 plays a critical role in establishing ACD in the epithelium. Production of phosphatidyl inositol triphosphate (PIP3 is localized to the apical side of basal cells. Asymmetric recruitment of atypical protein kinase C (aPKC and partitioning defective (PAR 3 is impaired in PDK1 conditional knockout (CKO epidermis. PDK1CKO keratinocytes do not undergo calcium-induced activation of aPKC or IGF1-induced activation of AKT and fail to differentiate. PDK1CKO epidermis shows decreased expression of Notch, a downstream effector of ACD, and restoration of Notch rescues defective expression of differentiation-induced Notch targets in vitro. We therefore propose that PDK1 signaling regulates the basal-to-suprabasal switch in developing epidermis by acting as both an activator and organizer of ACD and the Notch-dependent differentiation program.

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

Science.gov (United States)

Van de Werve, G; Hers, H G

1979-01-15

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

Fate of bulk organic matter, nitrogen, and pharmaceutically active compounds in batch experiments simulating soil aquifer treatment (SAT) using primary effluent

KAUST Repository

Abel, Chol D T

2013-06-30

Reduction of bulk organic matter, nitrogen, and pharmaceutically active compounds from primary effluent during managed aquifer recharge was investigated using laboratory-scale batch reactors. Biologically stable batch reactors were spiked with different concentrations of sodium azide to inhibit biological activity and probe the effect of microbial activity on attenuation of various pollutants of concern. The experimental results obtained revealed that removal of dissolved organic carbon correlated with active microbial biomass. Furthermore, addition of 2 mM of sodium azide affected nitrite-oxidizing bacteria leading to accumulation of nitrite-nitrogen in the reactors while an ammonium-nitrogen reduction of 95.5 % was achieved. Removal efficiencies of the hydrophilic neutral compounds phenacetin, paracetamol, and caffeine were independent of the extent of the active microbial biomass and were >90 % in all reactors, whereas removal of pentoxifylline was dependent on the biological stability of the reactor. However, hydrophobic ionic compounds exhibited removal efficiency >80 % in batch reactors with the highest biological activity as evidenced by high concentration of adenosine triphosphate. © 2013 Springer Science+Business Media Dordrecht.

Fate of bulk organic matter, nitrogen, and pharmaceutically active compounds in batch experiments simulating soil aquifer treatment (SAT) using primary effluent

KAUST Repository

Abel, Chol D T; Sharma, Saroj K.; Maeng, Sungkyu; Magic-Knezev, Aleksandra; Kennedy, Maria Dolores; Amy, Gary L.

2013-01-01

Reduction of bulk organic matter, nitrogen, and pharmaceutically active compounds from primary effluent during managed aquifer recharge was investigated using laboratory-scale batch reactors. Biologically stable batch reactors were spiked with different concentrations of sodium azide to inhibit biological activity and probe the effect of microbial activity on attenuation of various pollutants of concern. The experimental results obtained revealed that removal of dissolved organic carbon correlated with active microbial biomass. Furthermore, addition of 2 mM of sodium azide affected nitrite-oxidizing bacteria leading to accumulation of nitrite-nitrogen in the reactors while an ammonium-nitrogen reduction of 95.5 % was achieved. Removal efficiencies of the hydrophilic neutral compounds phenacetin, paracetamol, and caffeine were independent of the extent of the active microbial biomass and were >90 % in all reactors, whereas removal of pentoxifylline was dependent on the biological stability of the reactor. However, hydrophobic ionic compounds exhibited removal efficiency >80 % in batch reactors with the highest biological activity as evidenced by high concentration of adenosine triphosphate. © 2013 Springer Science+Business Media Dordrecht.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

KAUST Repository

Lissanu Deribe, Yonathan

2016-03-01

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.

Science.gov (United States)

Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B; Wu, Chia-Chin; Akdemir, Kadir C; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T; Welch, Heidi C E; Garraway, Levi A; Chin, Lynda

2016-03-01

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

KAUST Repository

Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B.; Wu, Chia-Chin; Akdemir, Kadir C.; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T.; Welch, Heidi C. E.; Garraway, Levi A.; Chin, Lynda

2016-01-01

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Effect of captan on the exonuclease activities of DNA polymerase I from E. coli and reverse transcriptase from avian myeloblastosis virus

International Nuclear Information System (INIS)

Freeman-Wittig, M.J.B.

1986-01-01

The DNA pol I polymerase activity is known to be inhibited by captan. When captan was tested for its ability to alter the exonuclease activity of DNA pol I, degradation was enhanced at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. By assaying the two exonuclease activities separately it was shown that the differential effect by captan was the result of a combined inhibition of the 3' → 5' exonuclease and enhancement of the 5' → 3' exonuclease. Studies employing [ 14 C] captan showed that the alterations in DNA pol I activities were a result of the irreversible binding of captan to the enzyme in a ratio of 1:1. The effect of captan on AMV reverse transcriptase RNase H activity was also studied. RNase H activity appeared to be more sensitive to captan than was the polymerase activity. Inhibition of the polymerase activity could be prevented by deoxynucleotide triphosphate and was increased by templateprimer. RNase H activity, which showed a sigmoidal relationship between activity and substrate concentration, decreased in V/sub max/ with no change in the Hill coefficient in the presence of captan

An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis.

Science.gov (United States)

Sarkar, Avijit; Sen, Rupashree; Saha, Piu; Ganguly, Sudipto; Mandal, Goutam; Chatterjee, Mitali

2008-05-01

An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

Science.gov (United States)

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Comparison of plate counts, Petrifilm, dipslides, and adenosine triphosphate bioluminescence for monitoring bacteria in cooling-tower waters.

Science.gov (United States)

Mueller, Sherry A; Anderson, James E; Kim, Byung R; Ball, James C

2009-04-01

Effective bacterial control in cooling-tower systems requires accurate and timely methods to count bacteria. Plate-count methods are difficult to implement on-site, because they are time- and labor-intensive and require sterile techniques. Several field-applicable methods (dipslides, Petrifilm, and adenosine triphosphate [ATP] bioluminescence) were compared with the plate count for two sample matrices--phosphate-buffered saline solution containing a pure culture of Pseudomonas fluorescens and cooling-tower water containing an undefined mixed bacterial culture. For the pure culture, (1) counts determined on nutrient agar and plate-count agar (PCA) media and expressed as colony-forming units (CFU) per milliliter were equivalent to those on R2A medium (p = 1.0 and p = 1.0, respectively); (2) Petrifilm counts were not significantly different from R2A plate counts (p = 0.99); (3) the dipslide counts were up to 2 log units higher than R2A plate counts, but this discrepancy was not statistically significant (p = 0.06); and (4) a discernable correlation (r2 = 0.67) existed between ATP readings and plate counts. For cooling-tower water samples (n = 62), (1) bacterial counts using R2A medium were higher (but not significant; p = 0.63) than nutrient agar and significantly higher than tryptone-glucose yeast extract (TGE; p = 0.03) and PCA (p < 0.001); (2) Petrifilm counts were significantly lower than nutrient agar or R2A (p = 0.02 and p < 0.001, respectively), but not statistically different from TGE, PCA, and dipslides (p = 0.55, p = 0.69, and p = 0.91, respectively); (3) the dipslide method yielded bacteria counts 1 to 3 log units lower than nutrient agar and R2A (p < 0.001), but was not significantly different from Petrifilm (p = 0.91), PCA (p = 1.00) or TGE (p = 0.07); (4) the differences between dipslides and the other methods became greater with a 6-day incubation time; and (5) the correlation between ATP readings and plate counts varied from system to system, was poor

Adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided platinum-based 2-drug chemotherapy for unresectable nonsmall-cell lung cancer.

Science.gov (United States)

Moon, Yong Wha; Choi, Sung Ho; Kim, Yong Tai; Sohn, Joo Hyuk; Chang, Joon; Kim, Se Kyu; Park, Moo Suk; Chung, Kyung Young; Lee, Hyoun Ju; Kim, Joo-Hang

2007-05-01

The study investigated correlations between adenosine triphosphate / chemotherapy response assay (ATP-CRA) and clinical outcomes after ATP-CRA-guided platinum-based chemotherapy for unresectable nonsmall-cell lung cancer (NSCLC). The authors performed an in vitro chemosensitivity test, ATP-CRA, to evaluate the chemosensitivities of anticancer drugs such as cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, and vinorelbine for chemonaive, unresectable NSCLC. The cell death rate was determined by measuring the intracellular ATP levels of drug-exposed cells compared with untreated controls. A sensitive drug was defined as a drug producing 30% or more reduction in ATP compared with untreated controls. Assay-guided platinum-based 2-drug chemotherapy was given to patients with pathologically confirmed NSCLC. Thirty-four patients were enrolled. Thirty tumor specimens were obtained by bronchoscopic biopsies and 4 obtained surgically. The median age was 61 years and 27 patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. The response rate was 43.8%. At a median follow-up period of 16.9 months, the median progression-free and overall survivals were 3.6 and 11.2 months, respectively. Patients were dichotomized into the platinum-sensitive (S; 20 patients) and resistant (R; 14 patients) groups. The positive/negative predictive values were 61.1% and 78.6% with a predictive accuracy of 68.8%. Although without significant differences in pretreatment parameters, the S-group showed better clinical response (P=.036), longer progression-free survival (P=.060), and longer overall survival (P=.025). Despite using bronchoscopic biopsied specimens, ATP-CRA and clinical outcomes correlated well after assay-guided platinum-based 2-drug chemotherapy for unresectable NSCLC. There was a favorable response and survival in the platinum-sensitive vs resistant groups. Copyright (c) 2007 American Cancer Society

Poly(adenosine 5'-diphosphate) ribose polymerase activation as a cause of metabolic dysfunction in critical illness.

Science.gov (United States)

Liaudet, Lucas

2002-03-01

Poly(adenosine 5'-diphosphate) ribose polymerase is a nuclear enzyme activated in response to genotoxic stress induced by a variety of DNA damaging agents. Several oxygen and nitrogen-centered free radicals, notably peroxynitrite, are strong inducers of DNA damage and poly(adenosine 5'-diphosphate) ribose polymerase activation in vitro and in vivo. Activation of this nuclear enzyme depletes the intracellular stores of its substrate nicotinamide adenine dinucleotide, slowing the rate of glycolysis, mitochondrial electron transport and adenosine triphosphate formation. This process triggers a severe energetic crisis within the cell, leading to acute cell dysfunction and cell necrosis. Poly(adenosine 5'-diphosphate) ribose polymerase also plays an important role in the regulation of inflammatory cascades, through a functional association with various transcription factors and transcription co-activators. Recent works identified this enzyme as a critical mediator of cellular metabolic dysfunction, inflammatory injury, and organ damage in conditions associated with overwhelming oxidative stress, including systemic inflammation, circulatory shock, and ischemia-reperfusion. Accordingly, pharmacological inhibitors of poly(adenosine 5'-diphosphate) ribose polymerase protect against cell death and tissue injury in such conditions, and may therefore represent novel therapeutic tools to limit multiple organ damage and dysfunction in critically ill patients.

Crystal structures of T. b. rhodesiense adenosine kinase complexed with inhibitor and activator: implications for catalysis and hyperactivation.

Directory of Open Access Journals (Sweden)

Sabine Kuettel

2011-05-01

Full Text Available BACKGROUND: The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP utilizing adenosine triphosphate (ATP as the preferred phosphoryl donor. METHODS AND FINDINGS: Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK: the structure of TbrAK in complex with the bisubstrate inhibitor P(1,P(5-di(adenosine-5'-pentaphosphate (AP5A at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]morpholine (compound 1 at 2.8 Å resolution. CONCLUSIONS: The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.

Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone

Directory of Open Access Journals (Sweden)

Charlotte Detremmerie

2017-07-01

Full Text Available The natural compound thymoquinone, extracted from Nigella sativa (black cumin, is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC producing inosine 3ʹ,5ʹ-cyclic monophosphate (cyclic IMP rather than guanosine 3ʹ,5ʹ-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

Increased Na+/K(+)-pump activity and adenosine triphosphate utilization after compound 48/80-induced histamine secretion from rat mast cells

DEFF Research Database (Denmark)

Johansen, Torben; Praetorius, Birger Hans

1994-01-01

-production were measured by the bioluminescence technique (firefly lantern) and by measurement of the lactate production under anaerobic conditions (antimycin A, oligomycin), respectively. There was an increased requirement for ATP after the secretory response associated with an increased activity of the Na...

Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I

International Nuclear Information System (INIS)

Bernges, F.; Holler, E.

1988-01-01

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in K/sub m/ values and a decrease in V/sub max/ values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [ 3 H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II)

New Biocatalyst with Multiple Enzymatic Activities for Treatment of Complex Food Wastewaters

Directory of Open Access Journals (Sweden)

Olga Senko

2008-01-01

Full Text Available The cells of filamentous fungus R. oryzae entrapped in the polyvinyl alcohol cryogelare capable of producing various extracellular hydrolytic enzymes (proteases, amylases, lipases and are used for the treatment of complex wastewaters of food industry. Five types of media simulating the wastewater of various food enterprises were treated under batch conditions for 600 h. Fats containing mostly residues of unsaturated fatty acids, as well as casein, glucose, sucrose, starch, soybean flour and various salts were the main components of the treated wastewaters. The immobilized cells concurrently possessed lipolytic, amylolytic and proteolytic activities. The level of each enzymatic activity depended on the wastewater content. The physiological state of immobilized cells was monitored by bioluminescent method. The intracellular adenosine triphosphate (ATP concentration determined in the granules with immobilized cells was high enough and almost constant for all the period of biocatalyst application confirming thereby the active metabolic state of the cells. The study of mechanical strength of biocatalyst granules allowed revealing the differences in the values of modulus of biocatalyst elasticity at the beginning and at the end of its use for the wastewater treatment. The decrease in chemical oxygen demand of the tested media after their processing by immobilized biocatalyst was 68–79 % for one working cycle.

The mechanism of phospholipase Cγ1 activation

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Paweł Krawczyk

2011-08-01

Full Text Available Phospholipase C is an enzyme which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5P2 into second messengers inositol-1,4,5-triphosphate (Ins(1,4,5P3 and diacylglycerol (DAG. These messengers then promote the activation of protein kinase C and release of Ca2 from intracellular stores, initiating numerous cellular events including proliferation, differentiation, signal transduction, endocytosis, cytoskeletal reorganization or activation of ion channels. There have been identified 14 isozymes of PLC among which PLCγ1 and PLCγ2 are of particular interest. PLC contains catalytic region XY and a few regulatory domains: PH, EF and C2. The most unique features of these two enzymes are the Src homology domains (SH2, SH3 and split PH domain within the catalytic barrel. PLC1 and PLCγ2 have an identical domain structure, but they differ in their function and occurrence. Phospholipase Cγ1 is expressed ubiquitously, especially in the brain, thymus and lungs.PLCγ1 can be activated by receptor tyrosine kinases (i.e.: PDGFR, EGFR, FGFR, Trk, as well as non-receptor protein kinases (Src, Syk, Tec or phosphatidic acid, tau protein and its analogue.The molecular mechanism of PLCγ1 activation includes membrane recruitment, phosphorylation, rearrangements and activation in the presence of growth factors.In reference to PLCγ1 regulation, a number of positive and negative modulators have been considered. The most important positive modulator is phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5P2. Protein kinase A and C, tyrosine phosphatases (SHP-1, PTP-1B and Cbl, Grb2, Jak2/PTP-1B complex proteins have been described as negative regulators of PLCγ1 activation.

Sequence Classification: 525310 [

Lifescience Database Archive (English)

Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|62181723|ref|YP_218140.1| dihydroneopte...rin aldolase, also has dihydroneopterin triphosphate 2'-epimerase activity || http://www.ncbi.nlm.nih.gov/protein/62181723 ...

Sequence Classification: 290568 [

Lifescience Database Archive (English)

Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|16130954|ref|NP_417530.1| dihydroneopte...rin aldolase, also has dihydroneopterin triphosphate 2'-epimerase activity || http://www.ncbi.nlm.nih.gov/protein/16130954 ...

Curcumin attenuates Cr(VI)-induced ascites and changes in the activity of aconitase and F(1)F(0) ATPase and the ATP content in rat liver mitochondria.

Science.gov (United States)

García-Niño, Wylly Ramsés; Zazueta, Cecilia; Tapia, Edilia; Pedraza-Chaverri, José

2014-11-01

Occupational and environmental exposure to potassium dichromate (K2Cr2O7), a hexavalent chromium compound, can result in liver damage associated with oxidative stress and mitochondrial dysfunction. The purpose of this study was to evaluate the effect of the antioxidant curcumin (400 mg/kg b.w.) on the K2Cr2O7-induced injury, with special emphasis on ascitic fluid accumulation and oxidative phosphorylation mitochondrial enzymes and the adenosine triphosphate (ATP) levels in isolated mitochondria from livers of rats treated with K2Cr2O7 (15 mg/kg b.w.). Thus, curcumin attenuated the ascites generation, prevented the decrease in the activities of aconitase and F1F0 ATPase, and maintained the ATP levels. The activity of complex II was not completely reestablished by curcumin, whereas complexes III and IV activities were unchanged. © 2014 Wiley Periodicals, Inc.

Allele specific hybridization using oligonucleotide probes of very high specific activity: Discrimination of the human β/sup A/ and β/sup S/-globin genes

International Nuclear Information System (INIS)

Studencki, A.B.; Wallace, R.B.

1984-01-01

The repair activity of E. coli DNA polymerase I (Klenow fragment) was used to prepare nonadecanucleotide hybridization probes which were complementary either to the normal human β-globin (β/sup A/) or to the sickle cell human β-globin (β/sup S/) gene. Template directed polymerization of highly radiolabeled α-/sup 32/P-deoxyribonucleoside triphosphates (3200, 5000 and/or 7800 Ci/mmol) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 - 2.0 x 10/sup 10/ dpm/μg. The extremely high specific activities of these probes made it possible to detect the β/sup A/ and β/sup S/ single copy gene sequences in as little as 1 μg of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states. This means that it was possible to detect 0.5 - 1.0 x 10/sup -18/ moles of a given single copy sequence

Antifungal Activity and Biochemical Response of Cuminic Acid against Phytophthora capsici Leonian.

Science.gov (United States)

Wang, Yong; Sun, Yang; Zhang, Ying; Zhang, Xing; Feng, Juntao

2016-06-11

Phytophthora blight of pepper caused by Phytophthora capsici Leonian is a destructive disease throughout the world. Cuminic acid, extracted from the seed of Cuminum cyminum L., belongs to the benzoic acid chemical class. In this study, the sensitivity and biochemical response of P. capsici to cuminic acid was determined. The mean EC50 (50% effective concentration) values for cuminic acid in inhibiting mycelial growth and zoospore germination of the 54 studied P. capsici isolates were 14.54 ± 5.23 μg/mL and 6.97 ± 2.82 μg/mL, respectively. After treatment with cuminic acid, mycelial morphology, sporangium formation and mycelial respiration were significantly influenced; cell membrane permeability and DNA content increased markedly, but pyruvic acid content, adenosine triphosphate (ATP) content, and ATPase activity decreased compared with the untreated control. In pot experiments, cuminic acid exhibited both protective and curative activity. Importantly, POD and PAL activity of the pepper leaves increased after being treated with cuminic acid. These indicated that cuminic acid not only showed antifungal activity, but also could improve the defense capacity of the plants. All the results suggested that cuminic acid exhibits the potential to be developed as a new phytochemical fungicide, and this information increases our understanding of the mechanism of action of cuminic acid against Phytophthora capsici.

Differential effects of near-UV and visible light on active transport and other membrane processes in Escherichia coli

International Nuclear Information System (INIS)

Sprott, G.D.; Martin, W.G.; Schneider, N.

1976-01-01

The effects of monochromatic near-UV and visible light on active transport and several other membrane processes in Escherichia coli were investigated. Using mercury lines at 366, 405, 435, 546 and 578 nm, large differential effects were observed. Transport systems with photosensitive initial rates of uptake were classified into three groups on the basis of wavelength dependence. Three, and possibly four photosensitizers may be involved; three active under aerobic conditions and the fourth in the absence of oxygen. Respiration rate exhibited the same sensitivity as one of the groups, suggesting that the active uptake of member amino acids (e.g. glycine) is largely dependent on oxidation energy. The photosensitivity of glycine transport at 405 nm was not the result of inhibition of the membrane-bound Ca-Mg adenosine triphosphates as shown using an isogenic mutant strain. Cell viability was not affected at the highly active wavelength, 405 nm. Photoeffects on transport of α-methylglucoside were minimal at 366 and 405 nm, contrasting to most of the amino acids investigated. The relative photosensitivity of respiration and several amino acid transport systems depended on carbon source. (author)

Aeromonas caviae alters the cytosolic and mitochondrial creatine kinase activities in experimentally infected silver catfish: Impairment on renal bioenergetics.

Science.gov (United States)

Baldissera, Matheus D; Souza, Carine F; Júnior, Guerino B; Verdi, Camila Marina; Moreira, Karen L S; da Rocha, Maria Izabel U M; da Veiga, Marcelo L; Santos, Roberto C V; Vizzotto, Bruno S; Baldisserotto, Bernardo

2017-09-01

Cytosolic and mitochondrial creatine kinases (CK), through the creatine kinase-phosphocreatine (CK/PCr) system, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. However, the effects of bacterial infections on the kidney remain poorly understood and are limited only to histopathological analyses. Thus, the aim of this study was to investigate the involvement of cytosolic and mitochondrial CK activities in renal energetic homeostasis in silver catfish experimentally infected with Aeromonas caviae. Cytosolic CK activity decreased in infected animals, while mitochondrial CK activity increased compared to uninfected animals. Moreover, the activity of the sodium-potassium pump (Na + , K + -ATPase) decreased in infected animals compared to uninfected animals. Based on this evidence, it can be concluded that the inhibition of cytosolic CK activity by A. caviae causes an impairment on renal energy homeostasis through the depletion of adenosine triphosphate (ATP) levels. This contributes to the inhibition of Na + , K + -ATPase activity, although the mitochondrial CK activity acted in an attempt to restore the cytosolic ATP levels through a feedback mechanism. In summary, A. caviae infection causes a severe energetic imbalance in infected silver catfish, which may contribute to disease pathogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing

Energy Technology Data Exchange (ETDEWEB)

Liang, Ru-Ping; Qiu, Wei-Bin; Zhao, Hui-Fang; Xiang, Cai-Yun; Qiu, Jian-Ding, E-mail: jdqiu@ncu.edu.cn

2016-01-21

Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody–antigen interaction in the presence of casein kinase II (CK2) and adenosine 5′-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis. - Highlights: • We reported a novel ECL-RET biosensor for sensitive analysis of casein kinase II activity. • The successful ECL-RET between GQDs and GO could be established. • GQDs was employed for casein kinase II activity monitoring and inhibition assay. • Highly sensitive detection of CK2 activity and inhibition was achieved.

Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5',5'-triphosphate bridge.

Science.gov (United States)

Zytek, Malgorzata; Kowalska, Joanna; Lukaszewicz, Maciej; Wojtczak, Blazej A; Zuberek, Joanna; Ferenc-Mrozek, Aleksandra; Darzynkiewicz, Edward; Niedzwiecka, Anna; Jemielity, Jacek

2014-12-07

A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,β-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m3(2,2,7)GppNHpG) or β-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.

Sequence Classification: 155582 [

Lifescience Database Archive (English)

Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|33519541|ref|NP_878373.1| dihydrone...opterin aldolase, also has dihydroneopterin triphosphate 2'-epimerase activity || http://www.ncbi.nlm.nih.gov/protein/33519541 ...

DNA sensor cGAS-mediated immune recognition

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Pengyan Xia

2016-09-01

Full Text Available Abstract The host takes use of pattern recognition receptors (PRRs to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.

Metabolic factors affecting enhanced phosphorus uptake by activated sludge.

Science.gov (United States)

Boughton, W H; Gottfried, R J; Sinclair, N A; Yall, I

1971-10-01

Activated sludges obtained from the Rilling Road plant located at San Antonio, Tex., and from the Hyperion treatment plant located at Los Angeles, Calif., have the ability to remove all of the orthophosphate normally present in Tucson sewage within 3 hr after being added to the waste water. Phosphorus removal was independent of externally supplied sources of energy and ions, since orthophosphate and (32)P radioactivity were readily removed from tap water, glass-distilled water, and deionized water. Phosphorus uptake by Rilling sludge in the laboratory appears to be wholly biological, as it has an optimum pH range (7.7 to 9.7) and an optimum temperature range (24 to 37 C). It was inhibited by HgCl(2), iodoacetic acid, p-chloromercuribenzoic acid, NaN(3), and 2, 4-dinitrophenol (compounds that affect bacterial membrane permeability, sulfhydryl enzymes, and adenosine triphosphate synthesis). Uptake was inhibited by 1% NaCl but was not affected by 10(-3)m ethylenediaminetetraacetic acid disodium salt (a chelating agent for many metallic ions).

Hypoxia-induced tumor cell resistance is overcome by synergistic GAPDH-siRNA and chemotherapy co-delivered by long-circulating and cationic-interior liposomes

NARCIS (Netherlands)

Guan, J.; Sun, J.; Sun, F.; Lou, B.; Zhang, D.; Mashayekhi, V.; Sadeghi, N.; Storm, G.; Mastrobattista, E.; He, Z.

2017-01-01

Chemotherapeutic drug resistance of tumor cells under hypoxic conditions is caused by the inhibition of apoptosis by autophagy and drug efflux via adenosine triphosphate (ATP)-dependent transporter activation, among other factors. Here, we demonstrate that disrupting glyceraldehyde-3-phosphate

Endothelial RIG-I activation impairs endothelial function

International Nuclear Information System (INIS)

Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

2012-01-01

Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

Novel cellular bouton structure activated by ATP in the vascular wall of porcine retinal arterioles.

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Misfeldt, Mikkel Wölck; Aalkjaer, Christian; Simonsen, Ulf; Bek, Toke

2010-12-01

The retinal blood flow is regulated by the tone of resistance arterioles, which is influenced by purinergic compounds such as adenosine and adenosine 5'-triphosphate (ATP) released from the retinal tissue. However, it is unknown what cellular elements in the perivascular retina are responsible for the effect of purines on the tone of retinal arterioles. Porcine retinal arterioles were loaded with the calcium-sensitive fluorophore Oregon green. The vessels were mounted in a confocal myograph for simultaneous recordings of tone and calcium activity in cells of the vascular wall during stimulation with ATP and adenosine, with and without modifiers of these compounds. Additionally, immunohistochemistry was used to localize elements with calcium activity in the vascular wall. Hyperfluorescence indicating calcium activity was recorded in a population of abundant round boutons interspersed in a network of vimentin-positive processes located immediately external to the smooth muscle cell layer but internal to the perivascular glial cells. These structures showed calcium activity when the vessel was relaxed with ATP but not when it was relaxed with adenosine. Ryanodine reduced calcium activity in the boutons, whereas the ATP antagonist adenosine-5'-O-(α, β- methylene diphosphate) reduced calcium activity in both the boutons and vascular tone. The vasodilating effect of purines in porcine retinal tissue involves ATP-dependent calcium activity in a layer of cellular boutons located external to the vascular smooth muscle cells and internal to the perivascular glial cells.

Mapping the active site of vaccinia virus RNA triphosphatase

International Nuclear Information System (INIS)

Gong Chunling; Shuman, Stewart

2003-01-01

The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

Deciphering common recognition principles of nucleoside mono/di and tri-phosphates binding in diverse proteins via structural matching of their binding sites.

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Bhagavat, Raghu; Srinivasan, Narayanaswamy; Chandra, Nagasuma

2017-09-01

Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cyclopentenyl cytosine has biological and anti-tumour activity, but does not enhance the efficacy of gemcitabine and radiation in two animal tumour models

NARCIS (Netherlands)

van Bree, Chris; Barten-van Rijbroek, Angeliqué D.; Leen, René; Rodermond, Hans M.; van Kuilenburg, André B. P.; Kal, Henk B.

2009-01-01

Cyclopentenyl cytosine (CPEC), targetting the de novo biosynthesis of cytidine triphosphate (CTP), increases the cytotoxicity of gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) alone and in combination with irradiation in several human tumour cells in vitro. We investigated whether OPEC enhances

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

DEFF Research Database (Denmark)

Larsen, Dorthe Helena; Poinsignon, Catherine; Gudjonsson, Thorkell

2010-01-01

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate...

Detergent inhibited, heat labile nucleoside triphosphatase in cores of avian myeloblastosis virus

DEFF Research Database (Denmark)

Jensen, Kaj Frank

1978-01-01

Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis...

Event-Associated Oxygen Consumption Rate Increases ca. Five-Fold When Interictal Activity Transforms into Seizure-Like Events In Vitro

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Karl Schoknecht

2017-09-01

Full Text Available Neuronal injury due to seizures may result from a mismatch of energy demand and adenosine triphosphate (ATP synthesis. However, ATP demand and oxygen consumption rates have not been accurately determined, yet, for different patterns of epileptic activity, such as interictal and ictal events. We studied interictal-like and seizure-like epileptiform activity induced by the GABAA antagonist bicuculline alone, and with co-application of the M-current blocker XE-991, in rat hippocampal slices. Metabolic changes were investigated based on recording partial oxygen pressure, extracellular potassium concentration, and intracellular flavine adenine dinucleotide (FAD redox potential. Recorded data were used to calculate oxygen consumption and relative ATP consumption rates, cellular ATP depletion, and changes in FAD/FADH2 ratio by applying a reactive-diffusion and a two compartment metabolic model. Oxygen-consumption rates were ca. five times higher during seizure activity than interictal activity. Additionally, ATP consumption was higher during seizure activity (~94% above control than interictal activity (~15% above control. Modeling of FAD transients based on partial pressure of oxygen recordings confirmed increased energy demand during both seizure and interictal activity and predicted actual FAD autofluorescence recordings, thereby validating the model. Quantifying metabolic alterations during epileptiform activity has translational relevance as it may help to understand the contribution of energy supply and demand mismatches to seizure-induced injury.

Role of P2X7 on steroid synthesis in murine luteal cells

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Chunping Zhang

2016-03-01

Full Text Available The extracellular adenosine triphosphate (ATP regulates different cellular functions through activating purinergic receptors as a signalling molecule or neurotransmitter. P2X7 is highly expressed in murine small luteal cells. In this study, murine luteal cells were cultured in vitro and treated with P2X7 agonists – ATP and 2′(3′-O-(4-benzoyl-benzoyl-adenosine 50-triphosphate (BzATP and with P2X7 antagonist – brilliant blue G (BBG. We found that ATP and BzATP increased the production of progesterone and had no influence on the production of estradiol. BBG reversed the effect of BzATP and ATP. Further studies demonstrated that ATP and BzATP promoted the expression of CYP11A. These results revealed that P2X7 receptor activation is involved in the steroid synthesis in corpus luteum.

Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

Science.gov (United States)

Saeki, Ayumi; Suzuki, Toshihiko; Hasebe, Akira; Kamezaki, Ryousuke; Fujita, Mari; Nakazawa, Futoshi; Shibata, Ken-Ichiro

2017-03-01

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1β responsible for the development of the disease. However, the mechanism of IL-1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1β and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1β production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1β. The inhibitors for ATP receptor reduced IL-1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity. © 2016 John Wiley & Sons Ltd.

Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

Science.gov (United States)

Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

2008-11-01

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

ATP induces NO production in hippocampal neurons by P2X(7 receptor activation independent of glutamate signaling.

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Juan Francisco Codocedo

Full Text Available To assess the putative role of adenosine triphosphate (ATP upon nitric oxide (NO production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3'-O-(4-Benzoylbenzoyl ATP (Bz-ATP elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG or by N(ω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV, but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

Endothelial RIG-I activation impairs endothelial function

Energy Technology Data Exchange (ETDEWEB)

Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

2012-03-30

Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

Nutritional State-Dependent Ghrelin Activation of Vasopressin Neurons via Retrograde Trans-Neuronal–Glial Stimulation of Excitatory GABA Circuits

Science.gov (United States)

Haam, Juhee; Halmos, Katalin C.; Di, Shi

2014-01-01

Behavioral and physiological coupling between energy balance and fluid homeostasis is critical for survival. The orexigenic hormone ghrelin has been shown to stimulate the secretion of the osmoregulatory hormone vasopressin (VP), linking nutritional status to the control of blood osmolality, although the mechanism of this systemic crosstalk is unknown. Here, we show using electrophysiological recordings and calcium imaging in rat brain slices that ghrelin stimulates VP neurons in the hypothalamic paraventricular nucleus (PVN) in a nutritional state-dependent manner by activating an excitatory GABAergic synaptic input via a retrograde neuronal–glial circuit. In slices from fasted rats, ghrelin activation of a postsynaptic ghrelin receptor, the growth hormone secretagogue receptor type 1a (GHS-R1a), in VP neurons caused the dendritic release of VP, which stimulated astrocytes to release the gliotransmitter adenosine triphosphate (ATP). ATP activation of P2X receptors excited presynaptic GABA neurons to increase GABA release, which was excitatory to the VP neurons. This trans-neuronal–glial retrograde circuit activated by ghrelin provides an alternative means of stimulation of VP release and represents a novel mechanism of neuronal control by local neuronal–glial circuits. It also provides a potential cellular mechanism for the physiological integration of energy and fluid homeostasis. PMID:24790191

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

Science.gov (United States)

Flitney, F W; Singh, J

1980-07-01

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

How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

Science.gov (United States)

Dudev, Todor; Grauffel, Cédric; Lim, Carmay

2017-02-01

Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

Evidence for an RNA polymerization activity in axolotl and Xenopus egg extracts.

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Hélène Pelczar

2010-12-01

Full Text Available We have previously reported a post-transcriptional RNA amplification observed in vivo following injection of in vitro synthesized transcripts into axolotl oocytes, unfertilized (UFE or fertilized eggs. To further characterize this phenomenon, low speed extracts (LSE from axolotl and Xenopus UFE were prepared and tested in an RNA polymerization assay. The major conclusions are: i the amphibian extracts catalyze the incorporation of radioactive ribonucleotide in RNase but not DNase sensitive products showing that these products correspond to RNA; ii the phenomenon is resistant to α-amanitin, an inhibitor of RNA polymerases II and III and to cordycepin (3'dAMP, but sensitive to cordycepin 5'-triphosphate, an RNA elongation inhibitor, which supports the existence of an RNA polymerase activity different from polymerases II and III; the detection of radiolabelled RNA comigrating at the same length as the exogenous transcript added to the extracts allowed us to show that iii the RNA polymerization is not a 3' end labelling and that iv the radiolabelled RNA is single rather than double stranded. In vitro cell-free systems derived from amphibian UFE therefore validate our previous in vivo results hypothesizing the existence of an evolutionary conserved enzymatic activity with the properties of an RNA dependent RNA polymerase (RdRp.

Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds.

Science.gov (United States)

Marín-Aguilar, Fabiola; Pavillard, Luis E; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D

2017-01-29

Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases.

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

Science.gov (United States)

Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Phosphate Activation via Reduced Oxidation State Phosphorus (P. Mild Routes to Condensed-P Energy Currency Molecules

Directory of Open Access Journals (Sweden)

Claire R. Cousins

2013-07-01

Full Text Available The emergence of mechanisms for phosphorylating organic and inorganic molecules is a key step en route to the earliest living systems. At the heart of all contemporary biochemical systems reside reactive phosphorus (P molecules (such as adenosine triphosphate, ATP as energy currency molecules to drive endergonic metabolic processes and it has been proposed that a predecessor of such molecules could have been pyrophosphate [P2O74−; PPi(V]. Arguably the most geologically plausible route to PPi(V is dehydration of orthophosphate, Pi(V, normally a highly endergonic process in the absence of mechanisms for activating Pi(V. One possible solution to this problem recognizes the presence of reactive-P containing mineral phases, such as schreibersite [(Fe,Ni3P] within meteorites whose abundance on the early Earth would likely have been significant during a putative Hadean-Archean heavy bombardment. Here, we propose that the reduced oxidation state P-oxyacid, H-phosphite [HPO32−; Pi(III] could have activated Pi(V towards condensation via the intermediacy of the condensed oxyacid pyrophosphite [H2P2O52−; PPi(III]. We provide geologically plausible provenance for PPi(III along with evidence of its ability to activate Pi(V towards PPi(V formation under mild conditions (80 °C in water.

Optimisation of ATP determination in drinking water

DEFF Research Database (Denmark)

Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution,...... be separated from the water phase by filtration.......Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution...... and an Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...

The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.

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Mauney, Christopher H; Rogers, LeAnn C; Harris, Reuben S; Daniel, Larry W; Devarie-Baez, Nelmi O; Wu, Hanzhi; Furdui, Cristina M; Poole, Leslie B; Perrino, Fred W; Hollis, Thomas

2017-12-01

Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond "redox switch" that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.

Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

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Gao, Yuansheng; Chen, Zhengju; Leung, Susan W S; Vanhoutte, Paul M

2015-06-01

In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

The potential role of postsynaptic phospholipase C activity in synaptic facilitation and behavioral sensitization in Aplysia.

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Fulton, Daniel; Condro, Michael C; Pearce, Kaycey; Glanzman, David L

2008-07-01

Previous findings indicate that synaptic facilitation, a cellular mechanism underlying sensitization of the siphon withdrawal response (SWR) in Aplysia, depends on a cascade of postsynaptic events, including activation of inositol triphosphate (IP3) receptors and release of Ca2+ from postsynaptic intracellular stores. These findings suggest that phospholipase C (PLC), the enzyme that catalyzes IP3 formation, may play an important role in postsynaptic signaling during facilitation and learning in Aplysia. Using the PLC inhibitor U73122, we found that PLC activity is required for synaptic facilitation following a 10-min treatment with 5-HT, as measured at 20 min after 5-HT washout. Prior work has indicated that facilitation at this time is supported primarily by postsynaptic processes. To determine whether postsynaptic PLC activity is involved in 5-HT-mediated facilitatory actions, we examined the effect of U73122 on enhancement of the response of motor neurons isolated in cell culture to glutamate, the sensory neuron transmitter. A 10-min application of 5-HT induced persistent (>40 min) enhancement of glutamate-evoked potentials (Glu-EPs) recorded from isolated motor neurons, and this enhancement was blocked by U73122. Finally, we showed that injecting U73122 into intact animals before behavioral training impaired intermediate-term sensitization, indicating that PLC activity contributes to this form of nonassociative learning.

Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse

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Quan Li

2015-01-01

Full Text Available Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM and liquid cultured mycelia (TM in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM- and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP, antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5′-AMP-activated protein kinase (AMPK, and the expressions of peroxisome proliferator have activated receptor γ coactivator-1α (PGC-1α and phosphofructokinase-1 (PFK-1 in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases.

Ugonin U stimulates NLRP3 inflammasome activation and enhances inflammasome-mediated pathogen clearance

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Chun-Yu Chen

2017-04-01

Full Text Available The NOD-like receptor pyrin domain 3 (NLRP3 inflammasome contains Nod-like receptors, a subclass of pattern recognition receptors, suggesting that this complex has a prominent role in host defenses. Various structurally diverse stimulators activate the NLRP3 inflammasome through different signaling pathways. We previously reported that ugonin U (UgU, a natural flavonoid isolated from Helminthostachys zeylanica (L Hook, directly stimulates phospholipase C (PLC and triggers superoxide release in human neutrophils. In the present study, we showed that UgU induced NLRP3 inflammasome assembly and subsequent caspase-1 and interleukin (IL-1β processing in lipopolysaccharide-primed human monocytes. Moreover, UgU elicited mitochondrial superoxide generation in a dose-dependent manner, and a specific scavenger of mitochondrial reactive oxygen species (ROS diminished UgU-induced IL-1β and caspase-1 activation. UgU induced Ca2+ mobilization, which was inhibited by treatment with inhibitors of PLC or inositol triphosphate receptor (IP3R. Blocking Ca2+ mobilization, PLC, or IP3R diminished UgU-induced IL-1β release, caspase-1 activation, and mitochondrial ROS generation. These data demonstrated that UgU activated the NLPR3 inflammasome activation through Ca2+ mobilization and the production of mitochondrial ROS. We also demonstrated that UgU-dependent NLRP3 inflammasome activation enhanced the bactericidal function of human monocytes. The ability of UgU to stimulate human neutrophils and monocytes, both of which are professional phagocytes, and its capacity to activate the NLRP3 inflammasome, which is a promising molecular target for developing anti-infective medicine, indicate that UgU treatment should be considered as a possible novel therapy for treating infectious diseases.

Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate

International Nuclear Information System (INIS)

Xu, K.Y.; Kyte, J.

1989-01-01

An immunoadsorbent specific for the carboxy-terminal sequence -GAPER, which comprises residues 502-506 of the alpha-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase], was used to isolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H]acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys501, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E1 conformation or when the structure of the enzyme was changed from the E1 conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H]acetyl suggest that Lys501 from the alpha polypeptide is directly involved in binding ATP within the active site of (Na+ + K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodies raised against the sequence-GAPER and denatured or enzymically active (Na+ + K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the alpha-polypeptide, respectively

ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis.

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Fois, Giorgio; Winkelmann, Veronika Eva; Bareis, Lara; Staudenmaier, Laura; Hecht, Elena; Ziller, Charlotte; Ehinger, Konstantin; Schymeinsky, Jürgen; Kranz, Christine; Frick, Manfred

2018-02-05

Vesicular P2X 4 receptors are known to facilitate secretion and activation of pulmonary surfactant in the alveoli of the lungs. P2X 4 receptors are expressed in the membrane of lamellar bodies (LBs), large secretory lysosomes that store lung surfactant in alveolar type II epithelial cells, and become inserted into the plasma membrane after exocytosis. Subsequent activation of P2X 4 receptors by adenosine triphosphate (ATP) results in local fusion-activated cation entry (FACE), facilitating fusion pore dilation, surfactant secretion, and surfactant activation. Despite the importance of ATP in the alveoli, and hence lung function, the origin of ATP in the alveoli is still elusive. In this study, we demonstrate that ATP is stored within LBs themselves at a concentration of ∼1.9 mM. ATP is loaded into LBs by the vesicular nucleotide transporter but does not activate P2X 4 receptors because of the low intraluminal pH (5.5). However, the rise in intravesicular pH after opening of the exocytic fusion pore results in immediate activation of vesicular P2X 4 by vesicular ATP. Our data suggest a new model in which agonist (ATP) and receptor (P2X 4 ) are located in the same intracellular compartment (LB), protected from premature degradation (ATP) and activation (P2X 4 ), and ideally placed to ensure coordinated and timely receptor activation as soon as fusion occurs to facilitate surfactant secretion. © 2018 Fois et al.

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

Science.gov (United States)

Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

2018-07-05

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

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Javid-Majd, Farah; Yang, Dong [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States); Ioerger, Thomas R. [Department of Computer Science, Texas A& M University, College Station, Texas 77843-2128 (United States); Sacchettini, James C., E-mail: sacchett@tamu.edu [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States)

2008-06-01

The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

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Gregory Kohn

Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

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Priscila de Faria-Pinto

2010-07-01

Full Text Available In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80% of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14 after treatment (AT with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

Kinetic and photochemical studies and alteration of ultraviolet sensitivity of Escherichia coli thymidine kinase of halogenated allosteric regulators and substrate analogues

International Nuclear Information System (INIS)

Chen, M.S.; Prusoff, W.H.

1977-01-01

The effect of various halogenated and nonhalogenated allosteric effectors on the sensitivity of Escherichia coli thymidine kinase to ultraviolet radiations (uv,253.7 nm) was investigated. All naturally occurring dNTPs convert the monomeric form of the enzyme into the dimeric form which is less sensitive to uv inactivation. Whereas 5-iodo-2'-deoxycytidine triphosphate (IdCTP) and 5-iodo-2' deoxyuridine triphosphate (IdUTP) enhance the uv inactivation of the enzyme, 5-bromo-2'-deoxyuridine triphosphate and 5-bromo-2'-deoxycytidine triphosphate exert a protective effect similar to that produced by the corresponding naturally occurring effectors, dTTP and dCTP. The enhanced uv inactivation by IdUTP is prevented totally by dTTP, but only partially by dCTP or dThd, whereas the enhanced sensitization by IdCTP is prevented almost totally by dCTP, partially by dTTP, and not at all by dThd. The uv sensitization of thymidine kinase by IdCTP appears to be at the regulatory site since a maximum saturation effect is observed, and the concentration required to exert a 50% maximal uv sensitization is similar to its K/sub m/ for enhancement of catalytic activity. When the enzyme was irradiated in the presence of either [2- 14 C]IdUTP or [2- 14 C]IdUrd, zone sedimentation analysis in sucrose density gradients showed the sedimentation coefficient of the radioactive labeled proteins to be the same, 3.8 S. Hence uv irradiation of the effector-induced dimer resulted in not only dissociation to the monomer, but also complete loss of catalytic activity. The substitution of an azido group for the 5'-OH group of 5-iodo-, 5-bromo-, 5-chloro-, or 5-fluorodeoxyuridine greatly decreased their affinity for thymidine kinase, and in addition the kinetics of inhibition changed from a competitive to a noncompetitive pattern. The presence of the azido moiety in the 5' position of the halogenated nucleosides did not enhance the rate of uv inactivation of the enzyme

Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

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Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

2012-04-01

Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts

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Eun-Mi Noh

2016-01-01

Full Text Available Reactive oxygen species (ROS play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3 metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts.

Elevated plasma plasminogen activator inhibitor type-1 is an independent predictor of coronary microvascular dysfunction in hypertension

International Nuclear Information System (INIS)

Naya, Masanao; Tsukamoto, Takahiro; Inubushi, Masayuki; Morita, Koichi; Katoh, Chietsugu; Furumoto, Tomoo; Fujii, Satoshi; Tsutsui, Hiroyuki; Tamaki, Nagara

2007-01-01

Elevated plasma plasminogen activator inhibitor-1 (PAI-1) is related to cardiovascular events, but its role in subclinical coronary microvascular dysfunction remains unknown. Thus, in the present study it was investigated whether elevated plasma PAI-1 activity is associated with coronary microvascular dysfunction in hypertensive patients. Thirty patients with untreated essential hypertension and 10 age-matched healthy controls were studied prospectively. Myocardial blood flow (MBF) was measured by using 15 O-water positron emission tomography. Clinical variables associated with atherosclerosis (low-density lipoprotein-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, homeostasis model assessment (HOMA-IR), and PAI-1 activity) were assessed to determine their involvement in coronary microvascular dysfunction. Adenosine triphosphate (ATP)-induced hyperemic MBF and coronary flow reserve (CFR) were significantly lower in hypertensive patients than in healthy controls (ATP-induced MBF: 2.77±0.82 vs 3.49±0.71 ml·g -1 ·min -1 ; p<0.02 and CFR: 2.95±1.06 vs 4.25±0.69; p<0.001). By univariate analysis, CFR was positively correlated with HDL-cholesterol (r=0.46, p<0.02), and inversely with HOMA-IR (r=-0.39, p<0.05) and PAI-1 activity (r=-0.61, p<0.001). By multivariate analysis, elevated PAI-1 activity remained a significant independent determinant of diminished CFR. Elevated plasma PAI-1 activity was independently associated with coronary microvascular dysfunction, which suggests that plasma PAI-1 activity is an important clue linking hypofibrinolysis to the development of atherosclerosis. (author)

Melatonin Inhibits Neural Cell Apoptosis and Promotes Locomotor Recovery via Activation of the Wnt/β-Catenin Signaling Pathway After Spinal Cord Injury.

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Shen, Zhaoliang; Zhou, Zipeng; Gao, Shuang; Guo, Yue; Gao, Kai; Wang, Haoyu; Dang, Xiaoqian

2017-08-01

The spinal cord is highly sensitive to spinal cord injury (SCI) by external mechanical damage, resulting in irreversible neurological damage. Activation of the Wnt/β-catenin signaling pathway can effectively reduce apoptosis and protect against SCI. Melatonin, an indoleamine originally isolated from bovine pineal tissue, exerts neuroprotective effects after SCI through activation of the Wnt/β-catenin signaling pathway. In this study, we demonstrated that melatonin exhibited neuroprotective effects on neuronal apoptosis and supported functional recovery in a rat SCI model by activating the Wnt/β-catenin signaling pathway. We found that melatonin administration after SCI significantly upregulated the expression of low-density lipoprotein receptor related protein 6 phosphorylation (p-LRP-6), lymphoid enhancer factor-1 (LEF-1) and β-catenin protein in the spinal cord. Melatonin enhanced motor neuronal survival in the spinal cord ventral horn and improved the locomotor functions of rats after SCI. Melatonin administration after SCI also reduced the expression levels of Bax and cleaved caspase-3 in the spinal cord and the proportion of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cells, but increased the expression level of Bcl-2. These results suggest that melatonin attenuated SCI by activating the Wnt/β-catenin signaling pathway.

Modulation of Ionic Channel Function by Protein Phosphorylation

Science.gov (United States)

1992-11-12

were prepared from the cloned cDNAs using the SP6 RNA promoter/polymerase system (32) with capping accomplished by priming with cap analogues (33...triphosphate (ATP), cytidine triphosphate (CTP) and uridine triphosphate (UTP) [ct-32p]UTP at 80 Cilmmol; 0.1 mM GTP; 0.5 mM diguanosinetriphosphate; 200 g.g/ml...state NMR spectroscopy . J. Biomol. NMR 1:167-173. (1991). Tomich, J.M., A. Grove, T. Iwamoto, S. Marrer, M.S. Montal and M. Montal. Design principles

An adenylyl cyclase gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

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The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in som...

Activation of peripheral blood mononuclear cells by dengue virus infection depotentiates balapiravir.

Science.gov (United States)

Chen, Yen-Liang; Abdul Ghafar, Nahdiyah; Karuna, Ratna; Fu, Yilong; Lim, Siew Pheng; Schul, Wouter; Gu, Feng; Herve, Maxime; Yokohama, Fumiaki; Wang, Gang; Cerny, Daniela; Fink, Katja; Blasco, Francesca; Shi, Pei-Yong

2014-02-01

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC(50)). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC(50) was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.

Synthesis of tritium labelled nucleoside triphosphates by enzymatic phosphorylation

International Nuclear Information System (INIS)

Shen Decun; Ji Linzhen; Liao Sha

1986-01-01

[5- 3 H]UMP, [5- 3 H]CMP, [8- 3 H]AMP and [8- 3 H]GMP were prepared from 5BrUMP 5BrCMP 8BrAMP and 8BrGMP by catalytic halogentritium replacement at the same time. [5- 3 H]UTP, [5- 3 H]CTP, [8- 3 H]ATP and [8- 3 H]GTP were subsequently synthesized from [5- 3 H]UMP, [5- 3 H]CMP, [8- 3 H]AMP and [8- 3 H]GMP by enzymatic phosphorylation with the crude enzyme prepared from brewer's yeasts and purified by paper chromatography simultaneously. In addition, four kinds of tritium labelled nucleoside monophosphates and four kinds of tritium labelled nucleoside diphosphates were obtained as the by-products. The specific activity of these products is between 14-19 Ci/mmol and the radiochemical purity is more than 98%

Development of a human-specific B. thetaiotaomicron IMS/ATP assay for measuring viable human contamination in surface waters in Baja California, Mexico

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Immunomagnetic separation/adenosine triphosphate (IMS/ATP) assays utilize paramagnetic beads and target-specific antibodies to isolate target organisms. Following isolation, adenosine tri-phosphate (ATP) is extracted from the target population and quantified. An inversely-couple...

Intracellular Secretory Leukoprotease Inhibitor Modulates Inositol 1,4,5-Triphosphate Generation and Exerts an Anti-Inflammatory Effect on Neutrophils of Individuals with Cystic Fibrosis and Chronic Obstructive Pulmonary Disease

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Emer P. Reeves

2013-01-01

Full Text Available Secretory leukoprotease inhibitor (SLPI is an anti-inflammatory protein present in respiratory secretions. Whilst epithelial cell SLPI is extensively studied, neutrophil associated SLPI is poorly characterised. Neutrophil function including chemotaxis and degranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca2+ levels which is mediated by production of inositol 1,4,5-triphosphate (IP3 in response to G-protein-coupled receptor (GPCR stimuli. The aim of this study was to investigate the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease. Neutrophils were isolated from healthy controls (n=10, individuals with cystic fibrosis (CF (n=5 or chronic obstructive pulmonary disease (COPD (n=5. Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP and interleukin(IL-8 induced neutrophil chemotaxis (P<0.05 and decreased degranulation of matrix metalloprotease-9 (MMP-9, hCAP-18, and myeloperoxidase (MPO (P<0.05. The mechanism of inhibition involved modulation of cytosolic IP3 production and downstream Ca2+ flux. The described attenuation of Ca2+ flux was overcome by inclusion of exogenous IP3 in electropermeabilized cells. Inhibition of IP3 generation and Ca2+ flux by SLPI may represent a novel anti-inflammatory mechanism, thus strengthening the attractiveness of SLPI as a potential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF, non-CF bronchiectasis, and COPD.

ORF Alignment: NC_004337 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004337 gi|24113675 >1nbuA 1 118 5 120 2e-19 ... ref|NP_708185.1| D-erythro-7,8-dihydrone...opterin triphosphate epimerase [Shigella ... flexneri 2a str. 301] gb|AAN43892.1| ... D-erythro-7,8-dihydrone....1| ... D-erythro-7,8-dihydroneopterin triphosphate epimerase ... [...Shigella flexneri 2a str. 2457T] ref|NP_754732.1| ... D-erythro-7,8-dihydroneopterin triphosphate epi...merase ... [Escherichia coli CFT073] gb|AAP17710.1| ... D-erythro-7,8-dihydroneopterin triphos

ORF Alignment: NC_002655 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_002655 gi|15802850 >1nbuA 1 118 5 120 2e-19 ... ref|NP_708185.1| D-erythro-7,8-dihydrone...opterin triphosphate epimerase [Shigella ... flexneri 2a str. 301] gb|AAN43892.1| ... D-erythro-7,8-dihydrone....1| ... D-erythro-7,8-dihydroneopterin triphosphate epimerase ... [...Shigella flexneri 2a str. 2457T] ref|NP_754732.1| ... D-erythro-7,8-dihydroneopterin triphosphate epi...merase ... [Escherichia coli CFT073] gb|AAP17710.1| ... D-erythro-7,8-dihydroneopterin triphos

ORF Alignment: NC_004431 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004431 gi|26248692 >1nbuA 1 118 5 120 2e-19 ... ref|NP_708185.1| D-erythro-7,8-dihydrone...opterin triphosphate epimerase [Shigella ... flexneri 2a str. 301] gb|AAN43892.1| ... D-erythro-7,8-dihydrone....1| ... D-erythro-7,8-dihydroneopterin triphosphate epimerase ... [...Shigella flexneri 2a str. 2457T] ref|NP_754732.1| ... D-erythro-7,8-dihydroneopterin triphosphate epi...merase ... [Escherichia coli CFT073] gb|AAP17710.1| ... D-erythro-7,8-dihydroneopterin triphos

ORF Alignment: NC_004741 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_004741 gi|30063729 >1nbuA 1 118 5 120 2e-19 ... ref|NP_708185.1| D-erythro-7,8-dihydrone...opterin triphosphate epimerase [Shigella ... flexneri 2a str. 301] gb|AAN43892.1| ... D-erythro-7,8-dihydrone....1| ... D-erythro-7,8-dihydroneopterin triphosphate epimerase ... [...Shigella flexneri 2a str. 2457T] ref|NP_754732.1| ... D-erythro-7,8-dihydroneopterin triphosphate epi...merase ... [Escherichia coli CFT073] gb|AAP17710.1| ... D-erythro-7,8-dihydroneopterin triphos

Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

Science.gov (United States)

Greenhouse, W V; Lehninger, A L

1977-11-01

Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.

DREAM plays an important role in platelet activation and thrombogenesis

Science.gov (United States)

Kim, Kyungho; Tseng, Alan; Barazia, Andrew; Italiano, Joseph E.

2017-01-01

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain responses. However, it is unknown whether DREAM is expressed in anucleate platelets and plays a role in thrombogenesis. By using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that both hematopoietic and nonhematopoietic cell DREAMs are required for platelet thrombus formation following laser-induced arteriolar injury. In a FeCl3-induced thrombosis model, we found that compared with wild-type (WT) control and nonhematopoietic DREAM knockout (KO) mice, DREAM KO control and hematopoietic DREAM KO mice showed a significant delay in time to occlusion. Tail bleeding time was prolonged in DREAM KO control mice, but not in WT or DREAM bone marrow chimeric mice. In vivo adoptive transfer experiments further indicated the importance of platelet DREAM in thrombogenesis. We found that DREAM deletion does not alter the ultrastructural features of platelets but significantly impairs platelet aggregation and adenosine triphosphate secretion induced by numerous agonists (collagen-related peptide, adenosine 5′-diphosphate, A23187, thrombin, or U46619). Biochemical studies revealed that platelet DREAM positively regulates phosphoinositide 3-kinase (PI3K) activity during platelet activation. Using DREAM-null platelets and PI3K isoform-specific inhibitors, we observed that platelet DREAM is important for α-granule secretion, Ca2+ mobilization, and aggregation through PI3K class Iβ (PI3K-Iβ). Genetic and pharmacological studies in human megakaryoblastic MEG-01 cells showed that DREAM is important for A23187-induced Ca2+ mobilization and its regulatory function requires Ca2+ binding and PI3K-Iβ activation. These results suggest that platelet DREAM regulates PI3K-Iβ activity and plays an important role during thrombus formation. PMID:27903531

Dose dependent activation of retinoic acid-inducible gene-I promotes both proliferation and apoptosis signals in human head and neck squamous cell carcinoma.

Directory of Open Access Journals (Sweden)

Jingzhou Hu

Full Text Available The retinoic-acid-inducible gene (RIG-like receptor (RLR family proteins are major pathogen reorganization receptors (PRR responsible for detection of viral RNA, which initiates antiviral response. Here, we evaluated the functional role of one RLR family member, RIG-I, in human head and neck squamous cell carcinoma (HNSCC. RIG-I is abundantly expressed both in poorly-differentiated primary cancer and lymph node metastasis, but not in normal adjacent tissues. Activation of RIG-I by transfection with low dose of 5'-triphosphate RNA (3p-RNA induces low levels of interferon and proinflammatory cytokines and promotes NF-κB- and Akt-dependent cell proliferation, migration and invasion. In contrast, activation of RIG-I by a high dose of 3p-RNA induces robust mitochondria-derived apoptosis accompanied by decreased activation of Akt, which is independent of the interferon and TNFα receptor, but can be rescued by over-expression of constitutively active Akt. Furthermore, co-immunoprecipitation experiments indicate that the CARD domain of RIG-I is essential for inducing apoptosis by interacting with caspase-9. Together, our results reveal a dual role of RIG-I in HNSCC through regulating activation of Akt, in which RIG-I activation by low-dose viral dsRNA increases host cell survival, whereas higher level of RIG-I activation leads to apoptosis. These findings highlight the therapeutic potential of dsRNA mediated RIG-I activation in the treatment of HNSCC.

E-ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy.

Science.gov (United States)

Bach, Barbara Charlotte; Leal, Daniela Bitencourt Rosa; Jaques, Jeandre Augusto dos Santos; Souza, Viviane do Carmo Gonçalves; Ruchel, Jader Betsch; Schlemmer, Karine Bizzi; Zanette, Régis Adriel; Hecktheuer, Pedro Abib; de Lima Pereira, Patrique; Casali, Emerson André; Alves, Sydney Hartz; Santurio, Janio Morais

2013-08-01

Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2-like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto-adenosine deaminase (E-ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E-ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E-ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E-ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy. Copyright © 2012 John Wiley & Sons, Ltd.

Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

Science.gov (United States)

Koli, Swati; Prakash, Atul; Choudhury, Soumen; Mandil, Rajesh; Garg, Satish K

2018-05-21

Adverse effects of mercury on female reproduction are reported; however, its effect on myogenic activity of uterus and mechanism thereof is obscure. Present study was undertaken to unravel the mechanistic pathways of mercuric chloride (HgCl 2 )-induced myometrial contraction in rats. Isometric tension in myometrial strips of rats following in vitro exposure to HgCl 2 was recorded using data acquisition system-based physiograph. HgCl 2 produced concentration-dependent (10 nM-100 μM) uterotonic effect which was significantly (p Graphical Abstract Graphical abstract depicting the mechanism of mercury-induced myometrial contraction in rats. M receptor: Muscarinic receptor; PIP2: phospho-inositol bisphosphate; PLC: phospholipase-C; DAG: diacyl glycerol; IP3: inositol triphosphate; IP3R: inositol triphosphate receptor; PKC; protein kinase-C; MLCP: myosin light chain phosphatise; MYPT: myosin phosphatase; SR: sarco-endoplasmic reticulum.

The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

Science.gov (United States)

Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

2006-01-01

myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

International Nuclear Information System (INIS)

Traynor-Kaplan, A.E.; Thompson, B.L.; Harris, A.L.; Taylor, P.; Omann, G.M.; Sklar, L.A.

1989-01-01

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation

Selective effects of charge on G protein activation by FSH-receptor residues 551-555 and 650-653.

Science.gov (United States)

Grasso, P; Deziel, M R; Reichert, L E

1995-01-01

Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.

On the use of X-ray absorption spectroscopy to elucidate the structure of lutetium adenosine mono- and triphosphate complexes.

Science.gov (United States)

Mostapha, S; Berthon, C; Fontaine-Vive, F; Gaysinski, M; Guérin, L; Guillaumont, D; Massi, L; Monfardini, I; Solari, P L; Thomas, O P; Charbonnel, M C; Den Auwer, C

2014-02-01

Although the physiological impact of the actinide elements as nuclear toxicants has been widely investigated for half a century, a description of their interactions with biological molecules remains limited. It is however of primary importance to better assess the determinants of actinide speciation in cells and more generally in living organisms to unravel the molecular processes underlying actinide transport and deposition in tissues. The biological pathways of this family of elements in case of accidental contamination or chronic natural exposure (in the case of uranium rich soils for instance) are therefore a crucial issue of public health and of societal impact. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, phosphate derivatives are considered as probable targets of these cations. Among them, nucleotides and in particular adenosine mono- (AMP) and triphosphate (ATP) nucleotides occur in more chemical reactions than any other compounds on the earth's surface, except water, and are therefore critical target molecules. In the present study, we are interested in trans-plutonium actinide elements, in particular americium and curium that are more rarely considered in environmental and bioaccumulation studies than early actinides like uranium, neptunium and plutonium. A first step in this strategy is to work with chemical analogues like lanthanides that are not radioactive and therefore allow extended physical chemical characterization to be conducted that are difficult to perform with radioactive materials. We describe herein the interaction of lutetium(III) with adenosine AMP and ATP. With AMP and ATP, insoluble amorphous compounds have been obtained with molar ratios of 1:2 and 1:1, respectively. With an excess of ATP, with 1:2 molar ratio, a soluble complex has been obtained. A combination of spectroscopic techniques (IR, NMR, ESI-MS, EXAFS) together with quantum

Superoxide anion radicals induce IGF-1 resistance through concomitant activation of PTP1B and PTEN

Science.gov (United States)

Singh, Karmveer; Maity, Pallab; Krug, Linda; Meyer, Patrick; Treiber, Nicolai; Lucas, Tanja; Basu, Abhijit; Kochanek, Stefan; Wlaschek, Meinhard; Geiger, Hartmut; Scharffetter-Kochanek, Karin

2015-01-01

The evolutionarily conserved IGF-1 signalling pathway is associated with longevity, metabolism, tissue homeostasis, and cancer progression. Its regulation relies on the delicate balance between activating kinases and suppressing phosphatases and is still not very well understood. We report here that IGF-1 signalling in vitro and in a murine ageing model in vivo is suppressed in response to accumulation of superoxide anions () in mitochondria, either by chemical inhibition of complex I or by genetic silencing of -dismutating mitochondrial Sod2. The -dependent suppression of IGF-1 signalling resulted in decreased proliferation of murine dermal fibroblasts, affected translation initiation factors and suppressed the expression of α1(I), α1(III), and α2(I) collagen, the hallmarks of skin ageing. Enhanced led to activation of the phosphatases PTP1B and PTEN, which via dephosphorylation of the IGF-1 receptor and phosphatidylinositol 3,4,5-triphosphate dampened IGF-1 signalling. Genetic and pharmacologic inhibition of PTP1B and PTEN abrogated -induced IGF-1 resistance and rescued the ageing skin phenotype. We thus identify previously unreported signature events with , PTP1B, and PTEN as promising targets for drug development to prevent IGF-1 resistance-related pathologies. PMID:25520316

Physiological response in the European flounder (Platichthys flesus) to variable salinity and oxygen conditions

DEFF Research Database (Denmark)

Lundgreen, Kim; Kiilerich, Pia; Tipsmark, Christian Kølbæk

2008-01-01

. Muscle water content was the same at all three salinities, indicating complete cell volume regulation. Gill Na+/K+-ATPase activity did not change with salinity, but hypoxia caused a 25 % decrease in branchial Na+/K+-ATPase activity at all three salinities. Furthermore, hypoxia induced a significant...... the erythrocytic nucleoside triphosphate content, a common mechanism for increasing blood O2 affinity. It is concluded that moderate hypoxia induced an energy saving decrease in branchial Na+/K+-ATPase activity, which did not compromise extracellular osmoregulation....

Factor C*, the specific initiation component of the mouse RNA polymerase I holoenzyme, is inactivated early in the transcription process.

OpenAIRE

Brun, R P; Ryan, K; Sollner-Webb, B

1994-01-01

Factor C* is the component of the RNA polymerase I holoenzyme (factor C) that allows specific transcriptional initiation on a factor D (SL1)- and UBF-activated rRNA gene promoter. The in vitro transcriptional capacity of a preincubated rDNA promoter complex becomes exhausted very rapidly upon initiation of transcription. This is due to the rapid depletion of C* activity. In contrast, C* activity is not unstable in the absence of transcription, even in the presence of nucleoside triphosphates ...

Induction of mitophagy-mediated antitumor activity with folate-appended methyl-β-cyclodextrin.

Science.gov (United States)

Kameyama, Kazuhisa; Motoyama, Keiichi; Tanaka, Nao; Yamashita, Yuki; Higashi, Taishi; Arima, Hidetoshi

2017-01-01

Mitophagy is the specific autophagic elimination system of mitochondria, which regulates cellular survival via the removal of damaged mitochondria. Recently, we revealed that folate-appended methyl-β-cyclodextrin (FA-M-β-CyD) provides selective antitumor activity in folate receptor-α (FR-α)-expressing cells by the induction of autophagy. In this study, to gain insight into the detailed mechanism of this antitumor activity, we focused on the induction of mitophagy by the treatment of FR-α-expressing tumor cells with FA-M-β-CyD. In contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered KB cells, human epithelial cells from a fatal cervical carcinoma (FR-α (+)) through FR-α-mediated endocytosis. The transmembrane potential of isolated mitochondria after treatment with FA-M-β-CyD was significantly elevated. In addition, FA-M-β-CyD lowered adenosine triphosphate (ATP) production and promoted reactive oxygen species production in KB cells (FR-α (+)). Importantly, FA-M-β-CyD enhanced light chain 3 (LC3) conversion (LC3-I to LC3-II) in KB cells (FR-α (+)) and induced PTEN-induced putative kinase 1 (PINK1) protein expression, which is involved in the induction of mitophagy. Furthermore, FA-M-β-CyD had potent antitumor activity in BALB/c nu/nu mice xenografted with KB cells (FR-α (+)) without any significant side effects. Taken together, these findings demonstrate that the autophagic cell death elicited by FA-M-β-CyD could be associated with mitophagy induced by an impaired mitochondrial function.

A quick look at biochemistry : Carbohydrate metabolism

NARCIS (Netherlands)

Dashty, Monireh

2013-01-01

In mammals, there are different metabolic pathways in cells that break down fuel molecules to transfer their energy into high energy compounds such as adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), reduced nicotinamide adenine dinucleotide (NADH2), reduced flavin adenine

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

Science.gov (United States)

Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

2016-09-01

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p ACM (p ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line

NARCIS (Netherlands)

Verschuur, A. C.; Brinkman, J.; van Gennip, A. H.; Leen, R.; Vet, R. J.; Evers, L. M.; Voûte, P. A.; van Kuilenburg, A. B.

2001-01-01

Cyclopentenyl cytosine (CPEC) is a nucleoside-analogue that decreases the concentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resulting in a decreased synthesis of RNA and DNA. Low concentrations of dCTP

Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

Directory of Open Access Journals (Sweden)

Tigran R Petrosyan

2016-01-01

Full Text Available The study aims to confirm the neuroregenerative effects of bacterial melanin (BM on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12 or unilateral rubrospinal tract transection at the cervical level (C3–4 (group II; n = 12. In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup and the remaining six rats were intramuscularly injected with saline (saline subgroup. Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian's calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations.

Directory of Open Access Journals (Sweden)

Haibo Yu

2007-02-01

Full Text Available Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP, the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such "mechanochemical coupling" in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [] is to propose the actual mechanism behind these "population shifts" and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide

Purinergic signaling pathways in endocrine system.

Science.gov (United States)

Bjelobaba, Ivana; Janjic, Marija M; Stojilkovic, Stanko S

2015-09-01

Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. Published by Elsevier B.V.

Purinergic Signaling Pathways in Endocrine System

Science.gov (United States)

Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

2015-01-01

Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. PMID:25960051

Regulation of DNA repair processes in mammalian cell

International Nuclear Information System (INIS)

Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

1992-01-01

A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance : Transgenic TK2, mtDNA, and Antiretrovirals

OpenAIRE

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK...

Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

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Anderson Ronald

2009-10-01

Full Text Available Abstract Background The role of protein kinase C (PKC in regulating the activity of phospholipase C (PLC in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM, was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM and staurosporine (400 nM. Methods Alterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3, and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively. Results Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase. Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils. Conclusion Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.

Analysis of the inhibitory effects of VP-16-213 (etoposide) and podophyllotoxin on thymidine transport and metabolism in Ehrlich ascites tumor cells in vitro

International Nuclear Information System (INIS)

Yalowich, J.C.; Goldman, I.D.

1984-01-01

Uptake of 3 H after exposure of cells to [ 3 H]-thymidine is characterized by a rapid initial velocity that approximates membrane transport followed by a slower rate of uptake that parallels the accumulation of phosphorylated derivatives of thymidine, primarily thymidine triphosphate, within the cell. The high rate of thymidine transport relative to thymidine metabolism to the triphosphate within the cell decreases as the extracellular nucleoside concentration is reduced due to a much greater decrease in membrane transport than the subsequent metabolic step. Hence, as extracellular thymidine is decreased, transport becomes increasingly rate limiting to metabolism within the cell. VP-16-213 (etoposide) or podophyllotoxin inhibits the initial uptake rate for thymidine and, as a consequence, inhibits the intracellular formation of thymidine triphosphate. When extracellular thymidine is high, inhibitory effects on transport are transient, and the net rate of thymidine triphosphate accumulation within drug-treated cells rapidly approaches a velocity comparable to that of control cells, indicating no direct VP-16-213 or podophyllotoxin effect on nucleoside and nucleotide phosphorylation. When extracellular thymidine is reduced so that transport is rate limiting to metabolism, the duration of the inhibitory effects of VP-16-213 on thymidine triphosphate formation is prolonged. A secondary effect of VP-16-213 becomes manifest beyond 10 min of incubation with [3H]thymidine with the virtual complete cessation of thymidine incorporation into the acid precipitate without any change in the thymidine triphosphate level. This late effect is not observed with podophyllotoxin and indicates a direct effect of VP-16-213 on DNA synthesis that is distinct from the earlier inhibitory effect on thymidine phosphorylation, which is secondary to membrane transport

Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

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Wu, Yang-Chang [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Lan, Yu-Hsuan [School of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Chang, Fang-Rong [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Wen [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

2013-02-01

Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

Characterization of hydroxyurea (HYU) S49 T lymphoma cells

International Nuclear Information System (INIS)

Albert, D.A.; Gudas, L.J.

1986-01-01

This paper tests the hypotheses that in vivo ribonucleotide reductase activity and consequent deoxyribonucleoside triphosphate production is rate-limited by the availability of M2 activity. The authors selected and characterized cell lines with variable resistance to hydroxyurea and comparing them with wild type S49 T-lymphoma cells. Ribonucleotide reductase assay was measured and in the process C 14-CDP was added in the final volume of assay mixture. It is shown that hydroxourea reversibly binds to the tyrosine radical of the M2 subunit of ribonucleotide reductase that is required for catalytic activity

A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

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Ma’ayan Israeli

2016-08-01

Full Text Available Edema Factor (EF, the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP, and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.

Research on garlic capsule and selenium-vitamin A, vitamin B, vitamin C applied in therapy of acute hepatocellular damage in a rat model

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Jacob Kehinde Akintunde

2015-10-01

Conclusions: Collectively, the results suggest that therapeutic dose of lisinopril elicits toxicity in male rats through induction of oxidative damage and depletion of cellular adenosine triphosphate. The reversal effects of GAR and SACE during lisinopril treatment suggest that these antioxidants may find clinical application in cellular damage involving ROS and adenosine triphosphate.

Investigation of tritium tracer in nucleic acid components by hydrogenolysis of corresponding precursors. 7. Preparation of tritiated uracil, uridine-5'-mono-, di- and triphosphates of high molar activity

Energy Technology Data Exchange (ETDEWEB)

Myasoedov, N F; Sidorov, G V; Kuznetsova, O B; Frank-Kamenetskaya, M D; Lazurkina, T Yu; Orlova, V A

1984-01-01

Preparation of (5,6-/sup 3/H) uracil by hydrogenolysis of 5-bromo-6-chlorouracil with molar activity (1.59-1.63)x10/sup 15/ Bg/mol has been described. Hydrogenolysis reaction kinetics is studied, effect of the reaction conditions on molar activity of dehalogenation product is considered. Using enzyme fraction, separated from the E.coli cells tritium-labelled preparations of UMP, UTP and UTP from (5.6-/sup 3/H) uracil are made. Kinetics of enzymatic reactions is studied and conditions, permitting to synthesize both UMP and UTP and a mixture of nucleotides of different degree of phosphorylation in approximately equal amounts are selected. Separation of nucleotides labelled with tritium is conducted using ion exchange chromatography on DEAE cellulose. Molar activity of nucleotides labelled with tritium equals molar activity of initial (5.6-/sup 3/H) uracil, and radiochemical purity constitutes more than 95%.

Chemical and thermal modulation of molecular motor activities

Science.gov (United States)

Hong, Weili

Molecular motors of kinesin and dynein families are responsible for various intracellular activities, from long distance movement of organelles, vesicles, protein complexes, and mRNAs to powering mitotic processes. They can take nanometer steps using chemical energy from the hydrolysis of ATP (adenosine triphosphate), and their dysfunction is involved in many neurodegenerative diseases that require long distance transport of cargos. Here I report on the study of the properties of molecular motors at a single-molecule level using optical trappings. I first studied the inhibition properties of kinesin motors by marine natural compound adociasulfates. I showed that adociasulfates compete with microtubules for binding to kinesins and thus inhibit kinesins' activity. Although adociasulfates are a strong inhibitor for all kinesin members, they show a much higher inhibition effect for conventional kinesins than for mitotic kinesins. Thus adociasulfates can be used to specifically inhibit conventional kinesins. By comparing the inhibition of kinesins by two structurally similar adociasulfates, one can see that the negatively charged sulfate residue of adociasulfates can be replaced by other negative residues and thus make it possible for adociasulfate-derived compounds to be more cell permeable. Kinesins and dyneins move cargos towards opposite directions along a microtubule. Cargos with both kinesins and dyneins attached often move bidirectionally due to undergoing a tug-of-war between the oppositely moving kinesin and dynein motors. Here I studied the effect of temperature on microtubule-based kinesin and dynein motor transport. While kinesins' and dyneins' velocities are closely matched above 15 °C, below this temperature the dyneins' velocity decreases much faster than the kinesins'. The kinesins' and dyneins' forces do not measurably change with temperature. The results suggest that temperature has significant effects on bidirectional transport and can be used to

Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

Science.gov (United States)

Hu, Yufang; Zhang, Qingqing; Xu, Lihua; Wang, Jiao; Rao, Jiajia; Guo, Zhiyong; Wang, Sui

2017-11-01

Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H 2 O 2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation

NARCIS (Netherlands)

Feng, Qian; Langereis, Martijn A; Olagnier, David; Chiang, Cindy; van de Winkel, Roel; van Essen, Peter; Zoll, Jan; Hiscott, John; van Kuppeveld, Frank J M

2014-01-01

Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by

Location of the redox-active thiols of ribonucleotide reductase: sequences similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

International Nuclear Information System (INIS)

Lin, A.N.I.; Ashley, G.W.; Stubbe, J.

1987-01-01

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1- 14 C]iodoacetamide. The dithiothreitol-reduce E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14 C. Sequencing of tryptic peptides shows that 2.8 equiv of 14 C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14 C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14 C. Sequencing of tryptic peptides shows that 1.4 equiv of 14 C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I

Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

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Yu-Ting Kao

2014-07-01

Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

P2X7 receptor activation ameliorates CA3 neuronal damage via a tumor necrosis factor-α-mediated pathway in the rat hippocampus following status epilepticus

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Ryu Hea Jin

2011-06-01

Full Text Available Abstract Background The release of tumor necrosis factor-α (TNF-α appears depend on the P2X7 receptor, a purinergic receptor. In the present study, we addressed the question of whether P2X7 receptor-mediated TNF-α regulation is involved in pathogenesis and outcome of status epilepticus (SE. Methods SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, 2',3'-O-(4-benzoylbenzoyl-adenosine 5'-triphosphate (BzATP, adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP, A-438079, or A-740003 prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunit phosphorylations. Results Following SE, P2X7 receptor agonist (BzATP infusion increased TNF-α immunoreactivity in dentate granule cells as compared with that in saline-infused animals. In addition, TNF-α immunoreactivity was readily apparent in the mossy fibers, while TNF-α immunoreactivity in CA1-3 pyramidal cells was unaltered. However, P2X7 receptor antagonist (OxATP-, A-438079, and A-740003 infusion reduced SE-induced TNF-α expression in dentate granule cells. In the CA3 region, BzATP infusion attenuated SE-induced neuronal damage, accompanied by enhancement of p65-Ser276 and p65-Ser311 NF-κB subunit phosphorylations. In contrast, OxATP-, A-438079, and A-740003 infusions increased SE-induced neuronal death. Soluble TNF p55 receptor (sTNFp55R, and cotreatment with BzATP and sTNFp55R infusion also increased SE-induced neuronal damage in CA3 region. However, OxATP-, sTNFp55R or BzATP+sTNFp55R infusions could not exacerbate SE-induced neuronal damages in the dentate gyrus and the CA1 region, as compared to BzATP infusion. Conclusions These findings suggest that TNF-α induction by P2X7 receptor activation may ameliorate SE-induced CA3 neuronal damage via enhancing NF-κB p65-Ser276 and p65-Ser311 phosphorylations.

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR

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Zheng Wang

2016-09-01

Full Text Available Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

Active-site modification of mammalian DNA polymerase β with pyridoxal 5'-phosphate: Mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

International Nuclear Information System (INIS)

Basu, A.; Kedar, P.; Wilson, S.H.; Modak, M.J.

1989-01-01

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase β. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [ 3 H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate

Ozone regeneration of granular activated carbon for trihalomethane control.

Science.gov (United States)

He, Xuexiang; Elkouz, Mark; Inyang, Mandu; Dickenson, Eric; Wert, Eric C

2017-03-15

Spatial and temporal variations of trihalomethanes (THMs) in distribution systems have challenged water treatment facilities to comply with disinfection byproduct rules. In this study, granular activated carbon (GAC) and modified GAC (i.e., Ag-GAC and TiO 2 -GAC) were used to treat chlorinated tap water containing CHCl 3 (15-21μg/L), CHBrCl 2 (13-16μg/L), CHBr 2 Cl (13-14μg/L), and CHBr 3 (3μg/L). Following breakthrough of dissolved organic carbon (DOC), GAC were regenerated using conventional and novel methods. GAC regeneration efficiency was assessed by measuring adsorptive (DOC, UV absorbance at 254nm, and THMs) and physical (surface area and pore volume) properties. Thermal regeneration resulted in a brief period of additional DOC adsorption (bed volume, BV, ∼6000), while ozone regeneration was ineffective regardless of the GAC type. THM adsorption was restored by either method (e.g., BV for ≥80% breakthrough, CHBr 3  ∼44,000>CHBr 2 Cl ∼35,000>CHBrCl 2  ∼31,000>CHCl 3  ∼7000). Cellular and attached adenosine triphosphate measurements illustrated the antimicrobial effects of Ag-GAC, which may have allowed for the extended THM adsorption compared to the other GAC types. The results illustrate that ozone regeneration may be a viable in-situ alternative for the adsorption of THMs during localized treatment in drinking water distribution systems. Published by Elsevier B.V.

Microbial dechlorination activity during and after chemical oxidant treatment

Energy Technology Data Exchange (ETDEWEB)

Doğan-Subaşı, Eylem [Flemish Institute for Technological Research (VITO), Separation and Conversion Technology, Boeretang 200, 2400 Mol (Belgium); Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Gent (Belgium); Bastiaens, Leen, E-mail: leen.bastiaens@vito.be [Flemish Institute for Technological Research (VITO), Separation and Conversion Technology, Boeretang 200, 2400 Mol (Belgium); Boon, Nico [Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Gent (Belgium); Dejonghe, Winnie [Flemish Institute for Technological Research (VITO), Separation and Conversion Technology, Boeretang 200, 2400 Mol (Belgium)

2013-11-15

Highlights: • Combined treatment was possible below 0.5 g/L of KMnO{sub 4} and 1 g/L of Na{sub 2}S{sub 2}O{sub 8}. • By-products SO{sub 4}{sup 2−} and MnO{sub 2(s)} had inhibitory effects on dehalogenating bacteria. • Oxidation reduction potential (ORP) was identified as a crucial parameter for recovery of oxidant exposed cells. • Bioaugmentation is a necessity at 0.5 g/L of KMnO{sub 4} and 1 g/L of Na{sub 2}S{sub 2}O{sub 8} and above. -- Abstract: Potassium permanganate (PM) and sodium persulfate (PS) are used in soil remediation, however, their compatibility with a coinciding or subsequent biotreatment is poorly understood. In this study, different concentrations of PM (0.005–2 g/L) and PS (0.01–4.52 g/L) were applied and their effects on the abundance, activity, and reactivation potential of a dechlorinating enrichment culture were investigated. Expression of the tceA, vcrA and 16S rRNA genes of Dehalococcoides spp. were detected at 0.005–0.01 g/L PM and 0.01–0.02 g/L PS. However, with 0.5–2 g/L PM and 1.13–4.52 g/L PS no gene expression was recorded, neither were indicator molecules for total cell activity (Adenosine triphosphate, ATP) detected. Dilution did not promote the reactivation of the microbial cells when the redox potential was above −100 mV. Similarly, inoculated cells did not dechlorinate trichloroethene (TCE) above −100 mV. When the redox potential was decreased to −300 mV and the reactors were bioaugmented for a second time, dechlorination activity recovered, but only in the reactors with 1.13 and 2.26 g/L PS. In conclusion, our results show that chemical oxidants can be combined with a biotreatment at concentrations below 0.5 g/L PM and 1 g/L PS.

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Effect of the exposure to suspended solids on the enzymatic activity in the bivalve Sinonovacula constricta

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Guojun Yang

2017-01-01

Full Text Available Aquatic animals are susceptible to sudden changes of their living environment but they adopt strategies to cope with adverse environmental challenges. Contamination by suspended solids, often associated with a dramatic change in the concentrations of important water-quality variables is a frequent occurrence in China's coastal waters and estuaries. Here we studied the impact of suspended solids on the activities of the antioxidant enzymes superoxide dismutase (SOD and catalase (CAT, as well as adenosine triphosphates (including Na+ K+-ATPase, Mg+ +-ATPase, Ca+ +-ATPase and H+ K+-ATPase in the gills and visceral mass tissues of the molluscan bivalve Sinonovacula constricta exposed (4, 8, 12, 16, 20, and 24 days to various concentrations of suspended solids. Our results showed that the antioxidant enzymes cooperated closely to effectively scavenge superoxide anion free radicals and H2O2 (which can ultimately inhibit gill activity through the modification of SOD and/or CAT enzymatic activities. ATPases activity (considered to be a sensitive indicator of toxicity could play an effective role in the maintenance of functional integrity of the plasma membranes as well as some other intracellular functions. After the exposure, a decrease in the Na+ K+-ATPase, Mg+ +-ATPase, and Ca+ +-ATPase activity of the gills was observed suggesting that they were inhibited by the treatments. These results also indicated that, from day 4 to day 16, exposure to high concentrations of suspended solids had an inhibitory effect on the activity of H+-K+-ATPase in the visceral mass of S. constricta. However, after a period of adaptation the H+-K+-ATPase activity was restored to original levels. Our results suggest that long-term exposure to high levels of suspended solids disturb osmoregulation, gastric acid secretion and digestion, cause oxidative damage, as a consequence of antioxidant enzymes inactivation which eventually damages the gills, affect the food intake

Biologically active filters - An advanced water treatment process for contaminants of emerging concern.

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Zhang, Shuangyi; Gitungo, Stephen W; Axe, Lisa; Raczko, Robert F; Dyksen, John E

2017-05-01

With the increasing concern of contaminants of emerging concern (CECs) in source water, this study examines the hypothesis that existing filters in water treatment plants can be converted to biologically active filters (BAFs) to treat these compounds. Removals through bench-scale BAFs were evaluated as a function of media, granular activated carbon (GAC) and dual media, empty bed contact time (EBCT), and pre-ozonation. For GAC BAFs, greater oxygen consumption, increased pH drop, and greater dissolved organic carbon removal normalized to adenosine triphosphate (ATP) were observed indicating increased microbial activity as compared to anthracite/sand dual media BAFs. ATP concentrations in the upper portion of the BAFs were as much as four times greater than the middle and lower portions of the dual media and 1.5 times greater in GAC. Sixteen CECs were spiked in the source water. At an EBCT of 18 min (min), GAC BAFs were highly effective with overall removals greater than 80% without pre-ozonation; exceptions included tri(2-chloroethyl) phosphate and iopromide. With a 10 min EBCT, the degree of CECs removal was reduced with less than half of the compounds removed at greater than 80%. The dual media BAFs showed limited CECs removal with only four compounds removed at greater than 80%, and 10 compounds were reduced by less than 50% with either EBCT. This study demonstrated that GAC BAFs with and without pre-ozonation are an effective and advanced technology for treating emerging contaminants. On the other hand, pre-ozonation is needed for dual media BAFs to remove CECs. The most cost effective operating conditions for dual media BAFs were a 10 min EBCT with the application of pre-ozonation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Implementation of anion-receptor macrocycles in supramolecular tandem assays for enzymes involving nucleotides as substrates, products, and cofactors.

Science.gov (United States)

Florea, Mara; Nau, Werner M

2010-03-07

A supramolecular tandem assay for direct continuous monitoring of nucleotide triphosphate-dependent enzymes such as potato apyrase is described. The underlying principle of the assay relies on the use of anion-receptor macrocycles in combination with fluorescent dyes as reporter pairs. A combinatorial approach was used to identify two complementary reporter pairs, i.e. an amino-gamma-cyclodextrin with 2-anilinonaphtalene-6-sulfonate (ANS) as dye (fluorescence enhancement factor of 17 upon complexation) and a polycationic cyclophane with 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS) as dye (fluorescence decrease by a factor of more than 2000), which allow the kinetic monitoring of potato apyrase activity at different ATP concentration ranges (microM and mM) with different types of photophysical responses (switch-ON and switch-OFF). Competitive fluorescence titrations revealed a differential binding of ATP (strongest competitor) versus ADP and AMP, which constitutes the prerequisite for monitoring enzymatic conversions (dephosphorylation or phosphorylation) involving nucleotides. The assay was tested for different enzyme and substrate concentrations and exploited for the screening of activating additives, namely divalent transition metal ions (Ni(2+), Mg(2+), Mn(2+), and Ca(2+)). The transferability of the assay could be demonstrated by monitoring the dephosphorylation of other nucleotide triphosphates (GTP, TTP, and CTP).

Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

Science.gov (United States)

Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

2016-10-01

Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic transition on the seizure-like neuronal activity by astrocytic calcium channel block

International Nuclear Information System (INIS)

Li, Jiajia; Wang, Rong; Du, Mengmeng; Tang, Jun; Wu, Ying

2016-01-01

The involvement of astrocytes in neuronal firing dynamics is becoming increasingly evident. In this study, we used a classical hippocampal tripartite synapse model consisting of soma-dendrite coupled neuron models and a Hodgkin–Huxley-like astrocyte model, to investigate the seizure-like firing in the somatic neuron induced by the over-expressed neuronal N-methyl-d-aspartate (NMDA) receptors. Based on this model, we further investigated the effect of the astrocytic channel block on the neuronal firing through a bifurcation analysis. Results show that blocking inositol-1,4,5-triphosphate(IP3)-dependent calcium channel in astrocytes efficiently suppresses the astrocytic calcium oscillation, which in turn suppresses the seizure-like firing in the neuron.

Musculus soleus of rats at physical activity and L-carnitine and creatine phosphate effect

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Irina A. Khutorskaya

2017-09-01

Full Text Available Introduction: The study of the effect of metabolic drugs on the histochemical characteristics of soleus muscle is relevant for solving the problem of providing the training process in Russia with non-doping drugs for safe correction of the consequences of intense physical activity in athletes. Materials and Methods: Dynamic physical activity in rats (n = 24 was simulated by swimming “to the limit” with weighting of 10 % of body weight (20 days, 1 time per day. The experimental animals were divided into four groups (6 animals each: № 1 – control, № 2 – swimming + isotonic NaCl solution, № 3 and № 4 – swimming + L-carnitine or creatine phosphate 100.0 mg/kg daily intraperitoneally. The object of the study was musculus soleus. Differentiation of muscle fibers was carried out by the intensity of histochemical activity of succinate dehydrogenase (SDG and alkaline stable adenosine triphosphate (ATP of myosin. The percentage of muscle fibers was evaluated and their diameter was defined by the direct morphometry. The obtained data were treated statistically by Student’s T-test. Results: Swimming of the animals “to the limit” do not affect the ratio of fibers with different phenotypes in the soleus muscle. This indicator is genetically determined and was not modified by L-carnitine and creatine phosphate. Dynamic physical activity promotes the development of hypertrophy of muscle fibers of various types. The investigated medicaments of the metabolic type either do not influence on the formation of exerciseinduced hypertrophy (predominantly creatine phosphate or reduce the intensity of the hypertrophic process (predominantly L-carnitine under dynamic physical activity. Discussion and Conclusions: The obtained data indicate L-carnitine and creatine phosphate do not have an anabolic effect. Taking into account the relevant data on ability of L-carnitine and creatine phosphate to effectively correct a negative effects of intensive

Repair replication in permeabilized Escherichia coli

International Nuclear Information System (INIS)

Masker, W.E.; Simon, T.J.; Hanawalt, P.C.

1975-01-01

We have examined the modes of DNA synthesis in Escherichia coli strains made permeable to nucleoside triphosphates by treatment with toluene. In this quasi in vitro system, polymerase-I-deficient mutants exhibit a nonconservative mode of synthesis with properties expected for the resynthesis step of excision-repair. This uv-stimulated DNA synthesis can be performed by either DNA polymerase II or III and it also requires the uvrA gene product. It requires the four deoxynucleoside triphosphates; but, in contrast to the semiconservative mode, the ATP requirement can be partially satisfied by other nucleoside triphosphates. The ATP-dependent recBC nuclease is not involved. The observed uv-stimulated mode of DNA synthesis may be part of an alternate excision-repair mechanism which supplements or complements DNA-polymerase-I-dependent repair in vivo

Method for enzyme synthesis of radioactive thymine 5'-deoxyribonucleotides

International Nuclear Information System (INIS)

Nejedly, Z.; Ekl, J.; Hybs, K.; Kolina, J.; Filip, J.; Votruba, I.; Skoda, J.

1978-01-01

The enzyme synthesis is described for thymidine-5'-monophosphate, thymidine-5'-diphosphate and thymidine-5'-triphosphate specifically or nonspecifically labelled with 14 C or 3 H. The anabolic transformation of radioactive thymine to radioactive thymine 5'-deoxyribonucleotides is catalyzed by the action of enzyme preparations separated from Escherichia coli bacteria. It is achieved by the action of nonpurified cell-free extracts on special auxotrophic mutants of the thymine-dependent Escherichia coli SPT - strain in the presence of deoxyriboso-1-phosphate and adenosine-5'-triphosphate. The radioactive thymidine-5'-monophosphate may further be phosphorylated. In reaction mixtures, radioactive thymine, deoxyriboso-1-phosphate and adenosine-5'-triphosphate are used in molar ratios of 1:1:2 to 1:10:100, the optimum molar ratio being 1:5:10. (B.S.)

Development of biomass in a drinking water granular active carbon (GAC) filter.

Science.gov (United States)

Velten, Silvana; Boller, Markus; Köster, Oliver; Helbing, Jakob; Weilenmann, Hans-Ulrich; Hammes, Frederik

2011-12-01

Indigenous bacteria are essential for the performance of drinking water biofilters, yet this biological component remains poorly characterized. In the present study we followed biofilm formation and development in a granular activated carbon (GAC) filter on pilot-scale during the first six months of operation. GAC particles were sampled from four different depths (10, 45, 80 and 115 cm) and attached biomass was measured with adenosine tri-phosphate (ATP) analysis. The attached biomass accumulated rapidly on the GAC particles throughout all levels in the filter during the first 90 days of operation and maintained a steady state afterward. Vertical gradients of biomass density and growth rates were observed during start-up and also in steady state. During steady state, biomass concentrations ranged between 0.8-1.83 x 10(-6) g ATP/g GAC in the filter, and 22% of the influent dissolved organic carbon (DOC) was removed. Concomitant biomass production was about 1.8 × 10(12) cells/m(2)h, which represents a yield of 1.26 × 10(6) cells/μg. The bacteria assimilated only about 3% of the removed carbon as biomass. At one point during the operational period, a natural 5-fold increase in the influent phytoplankton concentration occurred. As a result, influent assimilable organic carbon concentrations increased and suspended bacteria in the filter effluent increased 3-fold as the direct consequence of increased growth in the biofilter. This study shows that the combination of different analytical methods allows detailed quantification of the microbiological activity in drinking water biofilters. Copyright © 2011 Elsevier Ltd. All rights reserved.

Quantitative circumferential strain analysis using adenosine triphosphate-stress/rest 3-T tagged magnetic resonance to evaluate regional contractile dysfunction in ischemic heart disease

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Nakamura, Masashi, E-mail: m.nakamura1230@gmail.com [Department of Radiology, Ehime University Graduate School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295 (Japan); Kido, Tomoyuki [Department of Radiology, Saiseikai Matsuyama Hospital, Ehime 791-0295 (Japan); Kido, Teruhito; Tanabe, Yuki; Matsuda, Takuya; Nishiyama, Yoshiko; Miyagawa, Masao; Mochizuki, Teruhito [Department of Radiology, Ehime University Graduate School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295 (Japan)

2015-08-15

Highlights: • Infarcted segments could be differentiated from non-ischemic and ischemic segments with high sensitivity and specificity under at rest conditions. • The time-to-peak circumferential strain values in infarcted segments were more significantly delayed than those in non-ischemic and ischemic segments. • Both circumferential strain and circumferential systolic strain rate values under ATP-stress conditions were significantly lower in ischemic segments than in non-ischemic segments. • Subtracting stress and rest circumferential strain had a higher diagnostic capability for ischemia relative to only utilizing rest or ATP-stress circumferential strain values. • A circumferential strain analysis using tagged MR can quantitatively assess contractile dysfunction in ischemic and infarcted myocardium. - Abstract: Purpose: We evaluated whether a quantitative circumferential strain (CS) analysis using adenosine triphosphate (ATP)-stress/rest 3-T tagged magnetic resonance (MR) imaging can depict myocardial ischemia as contractile dysfunction during stress in patients with suspected coronary artery disease (CAD). We evaluated whether it can differentiate between non-ischemia, myocardial ischemia, and infarction. We assessed its diagnostic performance in comparison with ATP-stress myocardial perfusion MR and late gadolinium enhancement (LGE)-MR imaging. Methods: In 38 patients suspected of having CAD, myocardial segments were categorized as non-ischemic (n = 485), ischemic (n = 74), or infarcted (n = 49) from the results of perfusion MR and LGE-MR. The peak negative CS value, peak circumferential systolic strain rate (CSR), and time-to-peak CS were measured in 16 segments. Results: A cutoff value of −12.0% for CS at rest allowed differentiation between infarcted and other segments with a sensitivity of 79%, specificity of 76%, accuracy of 76%, and an area under the curve (AUC) of 0.81. Additionally, a cutoff value of 477.3 ms for time-to-peak CS at rest

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

International Nuclear Information System (INIS)

Yoon, Jung-Hoon; Qiu Junzhuan; Cai Sheng; Chen Yuan; Cheetham, Michael E.; Shen Binghui; Pfeifer, Gerd P.

2006-01-01

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease

PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

Science.gov (United States)

Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2015-10-15

Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

Science.gov (United States)

Taibi, N; Dupont, J; Bouguermouh, Z; Froment, P; Ramé, C; Anane, A; Amirat, Z; Khammar, F

2017-03-01

In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA polymerase of the killer virus of yeast

International Nuclear Information System (INIS)

Georgopoulos, D.E.; Leibowitz, M.J.

1984-01-01

The L/sub A/ and M double-stranded (ds) RNA segments of the cytoplasmically inherited killer virus of Saccharomyces cerevisiae are encapsidated in virions that contain a DNA-independent transcriptase activity. This enzyme catalyzes the synthesis of full-length (+) stranded copies of the genomic dsRNA segments, denoted l/sub A/ and m. The L/sub A/ dsRNA segment appears to encode the major capsid protein in which both dsRNA molecules are encapsidated, while M dsRNA encodes products responsible for the two killer phenotypes of toxin production and resistance to toxin. Proteins extracted from transcriptionally active virions fail to cross-react with antibody to yeast DNA-dependent RNA polymerases, suggesting that none of the subunits of the host cell polymerases are active in viral transcription. Sequence analysis of the in vitro transcripts reveals neither to be 3'-terminally polyadenylated, although m contains an apparent internal polyA-like tract. In the presence of any three ribonucleoside triphosphates (0.5 mM), the fourth ribonucleoside triphosphate shows an optimal rate of incorporation into transcript at a concentration of 20 μM. However, in a 3-hour reaction, the yield of a product RNA increases with the concentration of the limiting ribonucleotide up to 0.5 mM. Gel electrophoresis of the reaction products reveals that increasing the substrate concentration accelerates the appearance of radioactivity in full-length l/sub A/ and m transcripts

Suppression of the E. coli SOS response by dNTP pool changes.

Science.gov (United States)

Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J; Schaaper, Roel M

2015-04-30

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

An Analysis of Responses to Defibrotide in the Pulmonary Vascular Bed of the Cat.

Science.gov (United States)

Kaye, Alan D; Skonieczny, Brendan D; Kaye, Aaron J; Harris, Zoey I; Luk, Eric J

2016-01-01

Defibrotide is a polydisperse mixture of single-stranded oligonucleotides with many pharmacologic properties and multiple actions on the vascular endothelium. Responses to defibrotide and other vasodepressor agents were evaluated in the pulmonary vascular bed of the cat under conditions of controlled pulmonary blood flow and constant left atrial pressure. Lobar arterial pressure was increased to a high steady level with the thromboxane A2 analog U-46619. Under increased-tone conditions, defibrotide caused dose-dependent decreases in lobar arterial pressure without altering systemic arterial and left atrial pressures. Responses to defibrotide were significantly attenuated after the administration of the cyclooxygenase inhibitor sodium meclofenamate. Responses to defibrotide were also significantly attenuated after the administration of both the adenosine 1 and 2 receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine and 8-(3-chlorostyryl)caffeine. Responses to defibrotide were not altered after the administration of the vascular selective adenosine triphosphate-sensitive potassium channel blocker U-37883A, or after the administration of the nitric oxide synthase inhibitor L-N-(1-iminoethyl)-ornithine. These data show that defibrotide has significant vasodepressor activity in the pulmonary vascular bed of the cat. They also suggest that pulmonary vasodilator responses to defibrotide are partially dependent on both the activation of the cyclooxygenase enzyme and adenosine 1 and 2 receptor pathways and independent of the activation of adenosine triphosphate-sensitive potassium channels or the synthesis of nitric oxide in the pulmonary vascular bed of the cat.

Physical mechanisms of biological molecular motors

International Nuclear Information System (INIS)

Miller, John H. Jr.; Vajrala, Vijayanand; Infante, Hans L.; Claycomb, James R.; Palanisami, Akilan; Fang Jie; Mercier, George T.

2009-01-01

Biological motors generally fall into two categories: (1) those that convert chemical into mechanical energy via hydrolysis of a nucleoside triphosphate, usually adenosine triphosphate, regarded as life's chemical currency of energy and (2) membrane bound motors driven directly by an ion gradient and/or membrane potential. Here we argue that electrostatic interactions play a vital role for both types of motors and, therefore, the tools of physics can greatly contribute to understanding biological motors

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing

OpenAIRE

Kim, Jin S.; Nanfara, Michael T.; Chodavarapu, Sundari; Jin, Kyeong S.; Babu, Vignesh?M.?P.; Ghazy, Mohamed A.; Chung, Scisung; Kaguni, Jon M.; Sutton, Mark D.; Cho, Yunje

2017-01-01

Abstract Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda?sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda?? clam...

Multiple Mechanisms are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

Energy Technology Data Exchange (ETDEWEB)

Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.; Opresko, Lee; Coffey, Robert J.; Zangar, Richard C.; Wiley, H. S.

2008-11-14

REVIEW ENTIRE DOCUMENT AT: https://pnlweb.pnl.gov/projects/bsd/ERICA%20Manuscripts%20for%20Review/KD%20Rodland%20D7E80/HMEC_transactivation_ms01_15+Figs.pdf ABSTRACT: Using a single nontransformed strain of human mammary epithelial cells, we found that the ability of multiple growth factors and cytokines to induce ERK phosphorylation was dependent on EGFR activity. These included lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factoralpha. In contrast, hepatocyte growth factor could stimulate ERK phosphorylation independent of EGFR activity...

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

Science.gov (United States)

Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

2009-02-01

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

The 5HT(1A) receptor ligand, S15535, antagonises G-protein activation: a [35S]GTPgammaS and [3H]S15535 autoradiography study.

Science.gov (United States)

Newman-Tancredi, A; Rivet, J; Chaput, C; Touzard, M; Verrièle, L; Millan, M J

1999-11-19

4-(Benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) is a highly selective ligand at 5-HT(1A) receptors. The present study compared its autoradiographic labelling of rat brain sections with its functional actions, visualised by guanylyl-5'-[gamma-thio]-triphosphate ([35S]GTPgammaS) autoradiography, which affords a measure of G-protein activation. [3H]S15535 binding was highest in hippocampus, frontal cortex, entorhinal cortex, lateral septum, interpeduncular nucleus and dorsal raphe, consistent with specific labelling of 5-HT(1A) receptors. In functional studies, S15535 (10 microM) did not markedly stimulate G-protein activation in any brain region, but abolished the activation induced by the selective 5-HT(1A) agonist, (+)-8-hydroxy-dipropyl-aminotetralin ((+)-8-OH-DPAT, 1 microM), in structures enriched in [3H]S15535 labelling. S15535 did not block 5-HT-stimulated activation in caudate nucleus or substantia nigra, regions where (+)-8-OH-DPAT was ineffective and [3H]S15535 binding was absent. Interestingly, S15535 attenuated (+)-8-OH-DPAT and 5-HT-stimulated G-protein activation in dorsal raphe, a region in which S15535 is known to exhibit agonist properties in vivo [Lejeune, F., Millan, M.J., 1998. Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)(1A) receptors: WAY100,635-reversible actions of the highly selective ligands, flesinoxan and S15535. Synapse 30, 172-180.]. The present data show that (i) [3H]S15535 labels pre- and post-synaptic populations of 5-HT(1A) sites in rat brain sections, (ii) S15535 exhibits antagonist properties at post-synaptic 5-HT(1A) receptors in corticolimbic regions, and (iii) S15535 also attenuates agonist-stimulated G-protein activation at raphe-localised 5-HT(1A) receptors.

The history of N-methanocarbathymidine: the investigation of a conformational concept leads to the discovery of a potent and selective nucleoside antiviral agent.

Science.gov (United States)

Marquez, Victor E; Hughes, Stephen H; Sei, Shizuko; Agbaria, Riad

2006-09-01

Conformationally locked (North)-methanocarbathymidine (N-MCT) and (South)-methanocarbathymidine (S-MCT) have been used to investigate the conformational preferences of kinases and polymerases. The herpes kinases show a distinct bias for S-MCT, while DNA polymerases almost exclusively incorporate the North 5'-triphosphate (N-MCT-TP). Only N-MCT demonstrated potent antiviral activity against herpes simplex viruses (HSV-1 and 2) and Kaposi's sarcoma-associated herpesvirus (KSHV). The activity of N-MCT depends on its metabolic transformation to N-MCT-TP by the herpes kinases (HSV-tk or KSHV-tk), which catalyze the mono and diphosphorylation steps; cellular kinases generate the triphosphate. N-MCT at a dose of 5.6 mg/kg was totally protective for mice inoculated intranasally with HSV-1. Tumor cells that are not responsive to antiviral therapy became sensitive to N-MCT if the cells expressed HSV-tk. N-MCT given twice daily (100 mg/kg) for 7 days completely inhibited the growth of MC38 tumors derived from cells that express HSV-tk in mice while exhibiting no effect on tumors derived from non-transduced cells. After i.p. administration, N-MCT was rapidly absorbed and distributed in all organs examined with slow penetration into brain and testes. N-MCT-TP was also a potent inhibitor of HIV replication in human osteosarcoma (HOS) cells expressing HSV-tk.

Structural basis for the binding and incorporation of nucleotide analogs with L-stereochemistry by human DNA polymerase λ

OpenAIRE

Vyas, Rajan; Zahurancik, Walter J.; Suo, Zucai

2014-01-01

DNA polymerases are known to select against L-nucleotides, the enantiomers of natural D-nucleotides. However, the structural basis for D-stereoselectivity of a DNA polymerase has not been established, although two L-nucleoside analogs, lamivudine and emtricitabine, have been widely used as anti-HIV and anti-hepatitis B drugs. Here, we report ternary crystal structures of human DNA polymerase λ in complex with DNA and L-deoxycytidine 5′-triphosphate, or its analogs (the triphosphates of lamivu...

Physical mechanisms of biological molecular motors

Energy Technology Data Exchange (ETDEWEB)

Miller, John H. Jr. [Department of Physics and Texas Center for Superconductivity, University of Houston, 4800 Calhoun Road, Ste. 617 SR1 Houston, TX 77204-5005 (United States)], E-mail: jhmiller@uh.edu; Vajrala, Vijayanand; Infante, Hans L. [Department of Physics and Texas Center for Superconductivity, University of Houston, 4800 Calhoun Road, Ste. 617 SR1 Houston, TX 77204-5005 (United States); Claycomb, James R. [Department of Physics and Texas Center for Superconductivity, University of Houston, 4800 Calhoun Road, Ste. 617 SR1 Houston, TX 77204-5005 (United States); Department of Mathematics and Physics, Houston Baptist University, 7502 Fondren Road, Houston, TX 77074-3298 (United States); Palanisami, Akilan; Fang Jie; Mercier, George T. [Department of Physics and Texas Center for Superconductivity, University of Houston, 4800 Calhoun Road, Ste. 617 SR1 Houston, TX 77204-5005 (United States)

2009-03-01

Biological motors generally fall into two categories: (1) those that convert chemical into mechanical energy via hydrolysis of a nucleoside triphosphate, usually adenosine triphosphate, regarded as life's chemical currency of energy and (2) membrane bound motors driven directly by an ion gradient and/or membrane potential. Here we argue that electrostatic interactions play a vital role for both types of motors and, therefore, the tools of physics can greatly contribute to understanding biological motors.

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

International Nuclear Information System (INIS)

Ryu, S.H.; Lee, S.Y.; Lee, K.Y.; Rhee, S.G.

1987-01-01

Inositol 1,4,5-triphosphate (Ins-1,4,5-P 3 ) is an important second-messenger molecule that mobilizes Ca 2+ from intracellular stores in response to the occupancy of receptor by various Ca 2+ -mobilizing agonists. The fate of Ins-1,4,5-P 3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P 3 to Ins-1,3,4,5-P 4 , whereas the latter forms Ins-1,4-P 2 . Recent studies suggest that Ins-1,3,4,5-P 4 might modulate the entry of Ca 2+ from an extracellular source. In the current report, the authors describe the partial purification of the 3-kinase from the cytosolic fraction of bovine brain and studies of its catalytic properties. They found that the 3-kinase activity is significantly activated by the Ca 2+ /calmodulin complex. Therefore, they propose that Ca 2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P 3 forms a complex with calmodulin, and that the Ca 2+ /calmodulin complex stimulates the conversion of Ins-1,4,5-P 3 , and intracellular Ca 2+ mobilizer, to Ins-1,3,4,5-P 4 , an extracellular Ca 2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3- 32 P]Ins-1,3,4,5-P 4 and [γ- 32 P]ATP by thin-layer chromatography. Using this new assay method, they evaluated kinetic parameters (K/sub m/ for ATP = 40 μM, K/sub m/ for Ins-1,4,5-P 3 = 0.7 μM, K/sub i/ for ADP = 12 μM) and divalent cation specificity (Mg 2+ > > Mn 2+ > Ca 2+ ) for the 3-kinase

Induction of mitophagy-mediated antitumor activity with folate-appended methyl-β-cyclodextrin

Directory of Open Access Journals (Sweden)

Kameyama K

2017-04-01

Full Text Available Kazuhisa Kameyama,1,* Keiichi Motoyama,1,* Nao Tanaka,1 Yuki Yamashita,1 Taishi Higashi,1 Hidetoshi Arima1,2,* 1Department of Physical Pharmaceutics, Graduate School of Pharmaceutical Sciences, 2Program for Leading Graduate Schools “HIGO (Health Life Science: Interdisciplinary and Glocal Oriented Program,” Kumamoto University, Kumamoto, Japan *These authors contributed equally to this work Abstract: Mitophagy is the specific autophagic elimination system of mitochondria, which regulates cellular survival via the removal of damaged mitochondria. Recently, we revealed that folate-appended methyl-β-cyclodextrin (FA-M-β-CyD provides selective antitumor activity in folate receptor-α (FR-α-expressing cells by the induction of autophagy. In this study, to gain insight into the detailed mechanism of this antitumor activity, we focused on the induction of mitophagy by the treatment of FR-α-expressing tumor cells with FA-M-β-CyD. In contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered KB cells, human epithelial cells from a fatal cervical carcinoma (FR-α (+ through FR-α-mediated endocytosis. The transmembrane potential of isolated mitochondria after treatment with FA-M-β-CyD was significantly elevated. In addition, FA-M-β-CyD lowered adenosine triphosphate (ATP production and promoted reactive oxygen species production in KB cells (FR-α (+. Importantly, FA-M-β-CyD enhanced light chain 3 (LC3 conversion (LC3-I to LC3-II in KB cells (FR-α (+ and induced PTEN-induced putative kinase 1 (PINK1 protein expression, which is involved in the induction of mitophagy. Furthermore, FA-M-β-CyD had potent antitumor activity in BALB/c nu/nu mice xenografted with KB cells (FR-α (+ without any significant side effects. Taken together, these findings demonstrate that the autophagic cell death elicited by FA-M-β-CyD could be associated with mitophagy induced by an impaired mitochondrial function. Keywords: mitophagy, autophagy, folate receptor, methyl

Effects of Near-Infrared Laser on Neural Cell Activity

International Nuclear Information System (INIS)

Mochizuki-Oda, Noriko; Kataoka, Yosky; Yamada, Hisao; Awazu, Kunio

2004-01-01

Near-infrared laser has been used to relieve patients from various kinds of pain caused by postherpetic neuralgesia, myofascial dysfunction, surgical and traumatic wound, cancer, and rheumatoid arthritis. Clinically, He-Ne (λ=632.8 nm, 780 nm) and Ga-Al-As (805 ± 25 nm) lasers are used to irradiate trigger points or nerve ganglion. However the precise mechanisms of such biological actions of the laser have not yet been resolved. Since laser therapy is often effective to suppress the pain caused by hyperactive excitation of sensory neurons, interactions with laser light and neural cells are suggested. As neural excitation requires large amount of energy liberated from adenosine triphosphate (ATP), we examined the effect of 830-nm laser irradiation on the energy metabolism of the rat central nervous system and isolated mitochondria from brain. The diode laser was applied for 15 min with irradiance of 4.8 W/cm2 on a 2 mm-diameter spot at the brain surface. Tissue ATP content of the irradiated area in the cerebral cortex was 19% higher than that of the non-treated area (opposite side of the cortex), whereas the ADP content showed no significant difference. Irradiation at another wavelength (652 nm) had no effect on either ATP or ADP contents. The temperature of the brain tissue was increased 4.5-5.0 deg. C during the irradiation of both 830-nm and 652-nm laser light. Direct irradiation of the mitochondrial suspension did not show any wavelength-dependent acceleration of respiration rate nor ATP synthesis. These results suggest that the increase in tissue ATP content did not result from the thermal effect, but from specific effect of the laser operated at 830 nm. Electrophysiological studies showed the hyperpolarization of membrane potential of isolated neurons and decrease in membrane resistance with irradiation of the laser, suggesting an activation of potassium channels. Intracellular ATP is reported to regulate some kinds of potassium channels. Possible mechanisms

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

OpenAIRE

Garone, Caterina; Garc??a-D??az, Beatriz; Emmanuele, Valentina; L??pez Garc??a, Luis Carlos; Tadesse, Saba; Akman, Hasan O.; Tanji, Kurenai; Quinzii, Catarina M.; Hirano, Michio

2014-01-01

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 −/− ) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-...

Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

DEFF Research Database (Denmark)

Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille

2008-01-01

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha......Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme...

Three-dimensional structure of the large cytoplasmic H-4-H-5 loop of Na(+)/K(+)-ATPase deduced by restraint-based comparative modeling shows only one ATP binding site

Czech Academy of Sciences Publication Activity Database

Ettrich, Rüdiger; Melicherčík, M.; Teisinger, Jan; Ettrichová, Olga; Krumscheid, R.; Hofbauerová, Kateřina; Kvasnička, P.; Schoner, W.; Amler, Evžen

2001-01-01

Roč. 7, č. 6 (2001), s. 184-192 ISSN 0948-5023 R&D Projects: GA MŠk VS961410; GA ČR GA204/98/0468; GA AV ČR IAA7011801; GA ČR GA204/98/0416 Grant - others:IWTZ(DE) TSR-088-97; Volkswagen Foundation(DE) I/74679 Institutional research plan: CEZ:AV0Z5011922 Keywords : Sodium potassium adenosine triphosphate * tertiary structure * adenosine triphosphate binding site Subject RIV: BO - Biophysics Impact factor: 1.011, year: 2001

Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

Science.gov (United States)

Waisertreiger, Irina S.-R.; Menezes, Miriam R.; Randazzo, James; Pavlov, Youri I.

2010-01-01

Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA) hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs. PMID:20936128

Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

Directory of Open Access Journals (Sweden)

Irina S.-R. Waisertreiger

2010-01-01

Full Text Available Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP deoxynucleoside triphosphate and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs.

Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts

International Nuclear Information System (INIS)

Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

2014-01-01

Highlights: • Uniaxial stretching activates Ca 2+ signaling in human lung fibroblasts. • Stretch-induced intracellular Ca 2+ elevation is mainly via Ca 2+ influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca 2+ influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca 2+ ] i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca 2+ ] i . The stretch-induced [Ca 2+ ] i elevation was attenuated in Ca 2+ -free solution. In contrast, the increase of [Ca 2+ ] i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd 3+ , ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca 2+ ] i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca 2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP

Characterization of mitochondrial respiratory chain energetics in the vestibular nucleus complex.

Science.gov (United States)

Ashton, John C; Khalessi, Amirala; Kapoor, Mohit; Clarkson, Andrew; Sammut, Ivan A; Darlington, Cynthia L; Smith, Paul F

2005-04-01

Despite having very high neuronal firing rates, the VNC does not have unusually high mitochondrial activity in vitro. This study is the first in which functionally active mitochondria from the hindbrain have been isolated and characterized. Neurons in the vestibular nucleus complex (VNC) have exceptionally high spontaneous firing rates. Neuronal mitochondria generate adenosine triphosphate critical for maintaining the membrane potentials required for axon firing. We therefore hypothesized a high rate of mitochondrial activity in the VNC. To test this hypothesis, we compared mitochondrial activity in the VNC with mitochondrial activity from another area of the hindbrain, the cerebellum. Mitochondrial respiratory activity was assessed by measuring oxidative phosphorylation and mitochondrial respiratory enzyme complex activity. Assay results were not significantly different in the VNC compared to those obtained with the cerebellum or with rat brain mitochondria in previous studies.

Behavioral and biochemical characteristics of rats preferring ethanol or water

International Nuclear Information System (INIS)

Kulikova, O.G.; Borodkin, Y.S.; Razumovskaya, N.I.; Shabanov, P.D.; Sokolovskaya, N.E.

1985-01-01

Considering that learning and memory processes are largely determined by the intensity of RNA synthesis in specific brain structure, the authors study the relationship between learning ability of rats preferring ethanol or water and the level of RNA-synthesizing activity of brain cell nuclei. RNA-synthesizing activity of cell nuclei from cortical gray matter of the animals was determined one month after selection by measuring incorporation of deuterium-uridine triphosphate. The numerical results were subjected to statistical analysis by Student's test at P 0.05. It is shown that the altered behavior of animals preferring ethanol is evidently based on disturbed interaction between mediator and genetic structures of brain cells

The examination of urine samples for pathogenic microbes by the luciferase assay for ATP. 1: The effect of the presence of fungi, fungal like bacteria and kidney cells in urine samples

Science.gov (United States)

Bush, V. N.

1973-01-01

A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.

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

OpenAIRE

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

1993-01-01

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....

A quantitative assay for lysosomal acidification rates in human osteoclasts

DEFF Research Database (Denmark)

Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld

2011-01-01

The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... assay with respect to lysosomal acidification and assess whether it is a reliable test of a compound's ability to inhibit acidification. Investigated were the expression levels of the lysosomal acidification machinery, the activation of the assay by adenosine triphosphate, H(+) and Cl(-) dependency...

Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling.

Science.gov (United States)

Okajima, F; Tomura, H; Sho, K; Kimura, T; Sato, K; Im, D S; Akbar, M; Kondo, Y

1997-01-01

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.

Estimation of adenosine triphosphate utilization of rat mast cells during and after anaphylactic histamine secretion

DEFF Research Database (Denmark)

Johansen, Torben

1990-01-01

during the time period of histamine secretion and immediately after its completion. During secretion the additional ATP-utilization above the basal level of ATP-synthesis was 0.51 pmol/10(3) cells. 2.5 min after cell activation, the rate of additional ATP-utilization was 0.30 pmol/10(3) cells...

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Smith, C.A.; Hanawalt, P.C.

1978-01-01

The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [ 3 H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3 H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg 2+ -dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

Science.gov (United States)

Alfazari, Ali S; Al-Dabbagh, Bayan; Al-Dhaheri, Wafa; Taha, Mazen S; Chebli, Ahmad A; Fontagnier, Eva M; Koutoubi, Zaher; Kochiyi, Jose; Karam, Sherif M; Souid, Abdul-Kader

2015-01-01

AIM: To measure biochemical parameters in stomach biopsies and test their suitability as diagnostic biomarkers for gastritis and precancerous lesions. METHODS: Biopsies were obtained from the stomachs of two groups of patients (n = 40) undergoing fiber-optic endoscopy due to upper gastrointestinal symptoms. In the first group (n = 17), only the corpus region was examined. Biopsies were processed for microscopic examination and measurement of mitochondrial O2 consumption (cellular respiration), cellular adenosine triphosphate (ATP), glutathione (GSH), and caspase activity. In the second group of patients (n = 23), both corpus and antral regions were studied. Some biopsies were processed for microscopic examination, while the others were used for measurements of cellular respiration and GSH level. RESULTS: Microscopic examinations of gastric corpus biopsies from 17 patients revealed normal mucosae in 8 patients, superficial gastritis in 7 patients, and chronic atrophic gastritis in 1 patient. In patients with normal histology, the rate (mean ± SD) of cellular respiration was 0.17 ± 0.02 μmol/L O2 min-1 mg-1, ATP content was 487 ± 493 pmol/mg, and GSH was 469 ± 98 pmol/mg. Caspase activity was detected in 3 out of 8 specimens. The values of ATP and caspase activity were highly variable. The presence of superficial gastritis had insignificant effects on the measured biomarkers. In the patient with atrophic gastritis, cellular respiration was high and ATP was relatively low, suggesting uncoupling oxidative phosphorylation. In the second cohort of patients, the examined biopsies showed either normal or superficial gastritis. The rate of cellular respiration (O2. μmol/L min-1 mg-1) was slightly higher in the corpus than the antrum (0.18 ± 0.05 vs 0.15 ± 0.04, P = 0.019). The value of GSH was about the same in both tissues (310 ± 135 vs 322 ± 155, P = 0.692). CONCLUSION: The corpus mucosa was metabolically more active than the antrum tissue. The data in this

Neutrophil microparticle production and inflammasome activation by hyperglycemia due to cytoskeletal instability.

Science.gov (United States)

Thom, Stephen R; Bhopale, Veena M; Yu, Kevin; Huang, Weiliang; Kane, Maureen A; Margolis, David J

2017-11-03

Microparticles are lipid bilayer-enclosed vesicles produced by cells under oxidative stress. MP production is elevated in patients with diabetes, but the underlying cellular mechanisms are poorly understood. We hypothesized that raising glucose above the physiological level of 5.5 mm would stimulate leukocytes to produce MPs and activate the nucleotide-binding domain, leucine-rich repeat pyrin domain-containing 3 (NLRP3) inflammasome. We found that when incubated in buffer with up to 20 mm glucose, human and murine neutrophils, but not monocytes, generate progressively more MPs with high interleukin (IL)-1β content. Enhanced MP production required generation of reactive chemical species by mitochondria, NADPH oxidase, and type 2 nitric-oxide synthase (NOS-2) and resulted in S -nitrosylation of actin. Depleting cells of capon (C-terminal PDZ ligand of neuronal nitric-oxide synthase protein), apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain (ASC), or pro-IL-1β prevented the hyperglycemia-induced enhancement of reactive species production, MP generation, and IL-1β synthesis. Additional components required for these responses included inositol 1,3,5-triphosphate receptors, PKC, and enhancement of filamentous-actin turnover. Numerous proteins become localized to short filamentous actin in response to S -nitrosylation, including vasodilator-stimulated phosphoprotein, focal adhesion kinase, the membrane phospholipid translocation enzymes flippase and floppase, capon, NLRP3, and ASC. We conclude that an interdependent oxidative stress response to hyperglycemia perturbs neutrophil cytoskeletal stability leading to MP production and IL-1β synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantitative imaging of brain energy metabolisms and neuroenergetics using in vivo X-nuclear 2H, 17O and 31P MRS at ultra-high field.

Science.gov (United States)

Zhu, Xiao-Hong; Lu, Ming; Chen, Wei

2018-07-01

Brain energy metabolism relies predominantly on glucose and oxygen utilization to generate biochemical energy in the form of adenosine triphosphate (ATP). ATP is essential for maintaining basal electrophysiological activities in a resting brain and supporting evoked neuronal activity under an activated state. Studying complex neuroenergetic processes in the brain requires sophisticated neuroimaging techniques enabling noninvasive and quantitative assessment of cerebral energy metabolisms and quantification of metabolic rates. Recent state-of-the-art in vivo X-nuclear MRS techniques, including 2 H, 17 O and 31 P MRS have shown promise, especially at ultra-high fields, in the quest for understanding neuroenergetics and brain function using preclinical models and in human subjects under healthy and diseased conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

Tritium labelled nucleotides: Heterogeneous metal catalyzed exchange labelling of ATP with tritium gas

International Nuclear Information System (INIS)

Jaiswal, D.K.; Morimoto, H.; Williams, P.G.; Wemmer, D.E.

1991-09-01

Adenosine 5' triphosphate (ATP) in aqueous solution has been labeled by exchange with tritium gas in the presence of palladium oxide catalyst. Comparison with our experiments using Pd/BaSO 4 as the catalyst shows that we have obtained product with higher specific activity and improved chemical purity. 3 H NMR spectroscopy of the tritiated ATP shows labelling in both the C-8 and C-2 positions, and the integral ratio of these positions was found to vary from 3:1 to 1:1 under different reaction conditions. 5 refs., 1 fig., 2 tabs

Structure of tetrapotassium copper cyclo-triphosphate tetrahydrate

International Nuclear Information System (INIS)

Durif, A.; Averbuch-Pouchot, M.T.

1987-01-01

CuK 4 (P 3 O 9 ) 2 .4H 2 O, M r =765.84, monoclinic, P2 1 /a, a=8.510(5), b=14.303(8); c=8.487(5) A, β=96.51(2) 0 , V=1026(2) A 3 , Z=2, D x =2.478 Mg m -3 , λ(AgKα)=0.5608A, μ=1.272 mm -1 , F(000)=758, T=293 K, final R=0.028 for 2336 independent reflexions. The crystal structure is built up by double layers of KO n polyhedra alternating with layers of CuO 6 octahedra, both perpendicular to the c axis. The phosphoric anion P 3 O 9 is a trimeric ring. (orig.)

Complexing of lanthanides with adenosine-5'-triphosphate

International Nuclear Information System (INIS)

Svetlova, I.E.; Smirnova, N.S.; Dobrynina, N.A.; Martynenko, L.I.; Evseev, A.M.

1988-01-01

REE complexing with adenozine-5-triphosphoric acid in aqueous solutions at 25 deg C is studied by the method of pH metric titration using mathematical simulation. Ranges of existence are found, the composition is determined, stability constants of complexes of different composition are calculated

Disruption of Pyridine Nucleotide Redox Status During Oxidative Challenge at Normal and Low-Glucose States: Implications for Cellular Adenosine Triphosphate, Mitochondrial Respiratory Activity, and Reducing Capacity in Colon Epithelial Cells

Science.gov (United States)

Circu, Magdalena L.; Maloney, Ronald E.

2011-01-01

Abstract We recently demonstrated that menadione (MQ), a redox cycling quinone, mediated the loss of mitochondrial glutathione/glutathione disulfide redox balance. In this study, we showed that MQ significantly disrupted cellular pyridine nucleotide (NAD+/NADH, NADP+/NADPH) redox balance that compromised cellular ATP, mitochondrial respiratory activity, and NADPH-dependent reducing capacity in colonic epithelial cells, a scenario that was exaggerated by low glucose. In the cytosol, MQ induced NAD+ loss concurrent with increased NADP+ and NAD kinase activity, but decreased NADPH. In the mitochondria, NADH loss occurred in conjunction with increased nicotinamide nucleotide transhydrogenase activity and NADP+, and decreased NADPH. These results are consistent with cytosolic NAD+-to-NADP+ and mitochondrial NADH-to-NADPH shifts, but compromised NADPH availability. Thus, despite the sacrifice of NAD+/NADH in favor of NADPH generation, steady-state NADPH levels were not maintained during MQ challenge. Impairments of cellular bioenergetics were evidenced by ATP losses and increased mitochondrial O2 dependence of pyridine nucleotide oxidation–reduction; half-maximal oxidation (P50) was 10-fold higher in low glucose, which was lowered by glutamate or succinate supplementation. This exaggerated O2 dependence is consistent with increased O2 diversion to nonmitochondrial O2 consumption by MQ-semiquinone redox cycling secondary to decreased NADPH-dependent MQ detoxication at low glucose, a situation that was corrected by glucose-sparing mitochondrial substrates. Antioxid. Redox Signal. 14, 2151–2162. PMID:21083422

Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro

KAUST Repository

Mulaudzi, Takalani

2011-09-01

While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O 2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Metabolic consequences of DNA damage: The role of poly (ADP-ribose) polymerase as mediator of the suicide response

International Nuclear Information System (INIS)

Berger, N.A.; Berger, S.J.

1986-01-01

Recent studies show that DNA damage can produce rapid alterations in steady state levels of deoxynucleoside triphosphate pools, for example, MNNG or uv-irradiation cause rapid increases in dATP and dTTP pools without significant changes in dGTP or dCTP pools. In vitro, studies with purified eukaryotic DNA polymerases show that the frequency of nucleotide misincorporation was affected by alterations in relative concentrations of the deoxynucleoside triphosphates. Thus the alterations in dNTP pool sizes that occur consequent to DNA damage may contribute to an increased mutagenic frequency. Poly(ADP-ribose) polymerase mediated suicide mechanism may participate in the toxicity of adenosine deaminase deficiency and severe combined immune deficiency disease in humans. Individuals with this disease suffer severe lymphopenia due to the toxic effects of deoxyadenosine. The lymphocytotoxic effect of adenosine deaminase deficiency can be simulated in lymphocyte cell lines from normal individuals by incubating them with the adenosine deaminase inhibitor, deoxycoformycin. Incubation of such leukocytes with deoxycoformycin and deoxyadenosine results in the gradual accumulation of DNA strand breaks and the depletion of NAD + leading to cell death over a period of several days. This depletion of NAD and loss of cell viability were effectively blocked by nicotinamide or 3-amino benzamide. Thus, persistent activation of poly(ADP-ribose) polymerase by unrepaired or recurrent DNA strand breaks may activate the suicide mechanism of cell death. This study provides a basis for the interesting suggestion that treatment with nicotinamide could block the persistent activity of poly(ADP-ribose) polymerase and may help preserve lymphocyte function in patients with adenosine deaminase deficiency. 16 refs., 3 figs., 2 tabs

Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3-bromopyruvate.

Science.gov (United States)

Thangaraju, Muthusamy; Karunakaran, Senthil K; Itagaki, Shiro; Gopal, Elangovan; Elangovan, Selvakumar; Prasad, Puttur D; Ganapathy, Vadivel

2009-10-15

3-bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3-bromopyruvate. The transport of 3-bromopyruvate by sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na+)-coupled, electrogenic transporter for short-chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3-bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence-activated cell-sorting analysis and colony-formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis. 3-Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na+-coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3-bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3-bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3-bromopyruvate. 3-Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na+ gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3. Copyright (c) 2009 American Cancer Society.

Protection against the clastogenic effect of CIS-PLATINUM by WR-2721, ATP and Vitamin C

International Nuclear Information System (INIS)

Rupova, I.; Yagova, A.

1993-01-01

The mutagenic effect of antineoplastic drug Cis-Platinum has been assessed by the frequency of micronuclei in polychromatic erythrocytes (PE) in the mouse bone marrow at time intervals of 24, 48 and 73 h. The maximal clastogenic effect of 2 mg/kg of Cis-Platinum has been found at 24 h interval after treatment (3.6%). The protection against mutagenic activity of Cis-Platinum has been studied by pretreatment of the mice with the thiophosphate compound WR-2721, adenosine triphosphate (ATP) and Vitamin C alone or in combinations at the time of maximal response. The radioprotector WR-2721 exerts a high anti-mutagenic activity when applied prior to Cis-Platinum (2.2%; p 0.05). (author)

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

Science.gov (United States)

Miller, Justin M; Enemark, Eric J

2016-01-01

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

A rapid and highly selective method for the estimation of pyro-, tri- and orthophosphates.

Science.gov (United States)

Kamat, D R; Savant, V V; Sathyanarayana, D N

1995-03-01

A rapid, highly selective and simple method has been developed for the quantitative determination of pyro-, tri- and orthophosphates. The method is based on the formation of a solid complex of bis(ethylenediamine)cobalt(III) species with pyrophosphate at pH 4.2-4.3, with triphosphate at pH 2.0-2.1 and with orthophosphate at pH 8.2-8.6. The proposed method for pyro- and triphosphates differs from the available method, which is based on the formation of an adduct with tris(ethylenediamine)cobalt(III) species. The complexes have the composition [Co(en)(2)HP(2)O(7)]4H(2)O and [Co(en)(2)H(2)P(3)O(10)]2H(2)O, respectively. The precipitation is instantaneous and quantitative under the recommended optimum conditions giving 99.5% gravimetric yield in both cases. There is no interferences from orthophosphate, trimetaphosphate and pyrophosphate species in the triphosphate estimation up to 5% of each component. The efficacy of the method has been established by determining pyrophosphate and triphosphate contents in various matrices. In the case of orthophosphate, the proposed method differs from the available methods such as ammonium phosphomolybdate, vanadophosphomolybdate and quinoline phosphomolybdate, which are based on the formation of a precipitate, followed by either titrimetry or gravimetry. The precipitation is instantaneous and the method is simple. Under the recommended pH and other reaction conditions, gravimetric yields of 99.6-100% are obtainable. The method is applicable to orthophosphoric acid and a variety of phosphate salts.

Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

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Fatma Simsek-Duran

Full Text Available BACKGROUND: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus and mouse (Mus musculus ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. METHODOLOGY/PRINCIPAL FINDINGS: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. CONCLUSIONS/SIGNIFICANCE: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

Science.gov (United States)

Rhaese, H J; Hoch, J A; Groscurth, R

1977-03-01

To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

Intercellular odontoblast communication via ATP mediated by pannexin-1 channel and phospholipase C-coupled receptor activation.

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Masaki eSato

2015-11-01

Full Text Available Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected form rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s, we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP release channel (pannexin-1 inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated

Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi

Science.gov (United States)

2013-01-01

Background Ceramide accumulation is considered a contributing factor to neuronal dysfunction and damage. However, the underlying mechanisms that occur following ischemic insult are still unclear. Methods In the present study, we established cerebral ischemia models using four-vessel occlusion and oxygen-glucose deprivation methods. The hippocampus neural cells were subjected to immunohistochemistry and immunofluorescence staining for ceramide and neutral sphingomyelinase 2 (nSMase2) levels; immunoprecipitation and immunoblot analysis for nSMase2, receptor for activated C kinase 1 (RACK1), embryonic ectoderm development (EED), p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK expression; SMase assay for nSMase and acid sphingomyelinase (aSMase) activity; real-time reverse transcription polymerase chain reaction for cytokine expression; and Nissl, microtubule-associated protein 2 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Results We found considerable production of ceramide in astrocytes, but not in neurons, during early cerebral ischemia. This was accompanied by the induction of nSMase (but not aSMase) activity in the rat hippocampi. The inhibition of nSMase2 activity effectively reduced ceramide accumulation in astrocytes and alleviated neuronal damage to some extent. Meanwhile, the expression levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6, were found to be upregulated, which may have played an import role in neuronal damage mediated by the nSMase2/ceramide pathway. Although enhanced binding of nSMase2 with RACK1 and EED were also observed after cerebral ischemia, nSMase2 activity was not blocked by the TNF-α receptor inhibitor through RACK1/EED signaling. p38MAPK, but not protein kinase Cζ or protein phosphatase 2B, was able to induce nSMase2 activation after ischemia. p38MAPK can be induced by A2B adenosine

Emerging therapies for the management of decompensated heart failure: from bench to bedside.

Science.gov (United States)

deGoma, Emil M; Vagelos, Randall H; Fowler, Michael B; Ashley, Euan A

2006-12-19

While pharmaceutical innovation has been highly successful in reducing mortality in chronic heart failure, this has not been matched by similar success in decompensated heart failure syndromes. Despite outstanding issues over definitions and end points, we argue in this paper that an unprecedented wealth of pharmacologic innovation may soon transform the management of these challenging patients. Agents that target contractility, such as cardiac myosin activators and novel adenosine triphosphate-dependent transmembrane sodium-potassium pump inhibitors, provide inotropic support without arrhythmogenic increases in cytosolic calcium or side effects of more traditional agents. Adenosine receptor blockade may improve glomerular filtration and diuresis by exerting a direct beneficial effect on glomerular blood flow while vasopressin antagonists promote free water excretion without compromising renal function and may simultaneously inhibit myocardial remodeling. Urodilatin, the renally synthesized isoform of atrial natriuretic peptide, may improve pulmonary congestion via vasodilation and enhanced diuresis. Finally, metabolic modulators such as perhexiline may optimize myocardial energy utilization by shifting adenosine triphosphate production from free fatty acids to glucose, a unique and conceptually appealing approach to the management of heart failure. These advances allow optimism not only for the advancement of our understanding and management of decompensated heart failure syndromes but for the translational research effort in heart failure biology in general.

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

International Nuclear Information System (INIS)

Shoemaker, D.G.; Lauf, P.K.

1983-01-01

The interaction of the cardiac glycoside [ 3 H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [ 3 H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [ 3 H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [ 3 H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor

Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

Science.gov (United States)

Bitensky, Mark W.; Yoshida, Tatsuro

1997-01-01

Method using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4.degree. C. storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4.degree. C. for prolonged periods of time is achieved by removing oxygen therefrom at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate.

Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

2014-10-10

Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D

DEFF Research Database (Denmark)

Vinggaard, Anne Marie; Jensen, T.; Morgan, C.P.

1996-01-01

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techniques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extracted and partially...... purified on one column before easy detection of activity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5'-[¿-thio]triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ribosylation factor (Arf)]. In this paper we report...... that the use of didecanoyl phosphatidylcholine (C-PC) in mammalian PLD assays considerably increases the detection limit. C-PC was compared with the commonly used dipalmitoyl phosphatidylcholine (C-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and pig brain, and from...

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

Science.gov (United States)

Unciuleac, Mihaela-Carmen; Smith, Paul C; Shuman, Stewart

2016-05-15

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a

1H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla

International Nuclear Information System (INIS)

Stewart, J.M.M.; Grisham, C.M.

1988-01-01

1 H nuclear magnetic relaxation measurements have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH 3 ) 4 ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH 4 ) 4 ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase and that Mn 2+ bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na-K-ATPase. From the paramagnetic effect of Mn 2+ bound to the APTase on the longitudinal relaxation rates of the protons of Co(NH 3 ) 4 ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous 31 P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35 0 , and the conformation of the ribose ring is slightly N-type. The bound Mn 2+ lies above and in the plane of the adenine ring. The distances from Mn 2+ to N 6 and N 7 are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme

Metabolic Agents that Enhance ATP can Improve Cognitive Functioning: A Review of the Evidence for Glucose, Oxygen, Pyruvate, Creatine, and l-Carnitine

Directory of Open Access Journals (Sweden)

Lauren Owen

2011-08-01

Full Text Available Over the past four or five decades, there has been increasing interest in the neurochemical regulation of cognition. This field received considerable attention in the 1980s, with the identification of possible cognition enhancing agents or “smart drugs”. Even though many of the optimistic claims for some agents have proven premature, evidence suggests that several metabolic agents may prove to be effective in improving and preserving cognitive performance and may lead to better cognitive aging through the lifespan. Aging is characterized by a progressive deterioration in physiological functions and metabolic processes. There are a number of agents with the potential to improve metabolic activity. Research is now beginning to identify these various agents and delineate their potential usefulness for improving cognition in health and disease. This review provides a brief overview of the metabolic agents glucose, oxygen, pyruvate, creatine, and l-carnitine and their beneficial effects on cognitive function. These agents are directly responsible for generating ATP (adenosine triphosphate the main cellular currency of energy. The brain is the most metabolically active organ in the body and as such is particularly vulnerable to disruption of energy resources. Therefore interventions that sustain adenosine triphosphate (ATP levels may have importance for improving neuronal dysfunction and loss. Moreover, recently, it has been observed that environmental conditions and diet can affect transgenerational gene expression via epigenetic mechanisms. Metabolic agents might play a role in regulation of nutritional epigenetic effects. In summary, the reviewed metabolic agents represent a promising strategy for improving cognitive function and possibly slowing or preventing cognitive decline.

Necrosulfonamide Attenuates Spinal Cord Injury via Necroptosis Inhibition.

Science.gov (United States)

Wang, Yongxiang; Wang, Jingcheng; Wang, Hua; Feng, Xinmin; Tao, Yuping; Yang, Jiandong; Cai, Jun

2018-03-31

Spinal cord injury (SCI) is a serious trauma without efficient treatment currently. Necroptosis can be blocked post injury by special inhibitors. This study is to investigate the effects, mechanism, and potential benefit of necrosulfonamide (NSA) for SCI therapy. Pathologic condition was detected using hematoxylin-eosin staining on injured spinal cord and other major organs. Necroptosis-related factors-RIP1, RIP3, and MLKL-were detected using Western blot. Detections on mitochondrial functions such as adenosine triphosphate generation and activities of superoxide dismutase and caspase-3 were also performed. Finally, ethologic performance was detected using a 21-point open-field locomotion test. Reduced lesions and protected neurons were found in the injured spinal cord after treatment with NSA using hematoxylin-eosin staining for pathologic detection. No obvious toxicity on rat liver, kidney, heart, and spleen was detected. Rather than RIP1 and RIP3, MLKL was significantly inhibited by the NSA using Western blot detection. Adenosine triphosphate generation was obviously decreased post injury but slightly increased after the NSA treatment, especially 24 hours post injury. No significant changes were found on activities of superoxide dismutase and caspase-3 after the treatment of NSA. Ethologic performance was significantly improved using a 21-point, open-field locomotion test. Our research indicates NSA attenuates the spinal cord injury via necroptosis inhibition. It might be a potential and safe chemical benefit for SCI therapy. To our knowledge, this is the first study on the effects of NSA as treatment of traumatic SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+-ATPase Activity and K+/Na+ Homeostasis in Sweet Potato.

Science.gov (United States)

Yu, Yicheng; Wang, Aimin; Li, Xiang; Kou, Meng; Wang, Wenjun; Chen, Xianyang; Xu, Tao; Zhu, Mingku; Ma, Daifu; Li, Zongyun; Sun, Jian

2018-01-01

Melatonin (MT) is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [ Ipomoea batatas (L.) Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K + /Na + homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K + /Na + homeostasis in sweet potato seedlings as indicated by the low reduced K + content in tissues and low accumulation of Na + content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K + efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM) H + -ATPase activity and intracellular adenosine triphosphate (ATP) level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG) accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA) β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H + -ATPase activity, and K + /Na + homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H + -ATPase activity and K + /Na + homeostasis in sweet potato.

RNA polymerase activity of Ustilago maydis virus

Energy Technology Data Exchange (ETDEWEB)

Yie, S.W.

1986-01-01

Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

The advantage of channeling nucleotides for very processive functions [version 2; referees: 3 approved

Directory of Open Access Journals (Sweden)

Diana Zala

2017-07-01

Full Text Available Nucleoside triphosphate (NTPs, like ATP (adenosine 5’-triphosphate and GTP (guanosine 5’-triphosphate, have long been considered sufficiently concentrated and diffusible to fuel all cellular ATPases (adenosine triphosphatases and GTPases (guanosine triphosphatases in an energetically healthy cell without becoming limiting for function. However, increasing evidence for the importance of local ATP and GTP pools, synthesised in close proximity to ATP- or GTP-consuming reactions, has fundamentally challenged our view of energy metabolism. It has become evident that cellular energy metabolism occurs in many specialised ‘microcompartments’, where energy in the form of NTPs is transferred preferentially from NTP-generating modules directly to NTP-consuming modules. Such energy channeling occurs when diffusion through the cytosol is limited, where these modules are physically close and, in particular, if the NTP-consuming reaction has a very high turnover, i.e. is very processive. Here, we summarise the evidence for these conclusions and describe new insights into the physiological importance and molecular mechanisms of energy channeling gained from recent studies. In particular, we describe the role of glycolytic enzymes for axonal vesicle transport and nucleoside diphosphate kinases for the functions of dynamins and dynamin-related GTPases.

Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.

Science.gov (United States)

Vázquez-Cuevas, Francisco G; Martínez-Ramírez, Angélica S; Robles-Martínez, Leticia; Garay, Edith; García-Carrancá, Alejandro; Pérez-Montiel, Delia; Castañeda-García, Carolina; Arellano, Rogelio O

2014-11-01

P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability. © 2014 Wiley Periodicals, Inc.

Aging and defense against generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA

DEFF Research Database (Denmark)

Mikkelsen, Lone; Bialkowski, Karol; Risom, Lotte

2009-01-01

damaged DNA and the activity of the DNA repair system and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxodGTPase) activity in liver and lung tissue from mice at 10-100 weeks of age. The level of 8-oxodG increased with age, whereas the level of formamidopyrimidine DNA...... glycosylase sites was unaltered. The enzyme activity toward single oxygen-induced DNA damage and mRNA expression levels of Ercc1, Neil1, and Ogg1 remained unaltered with age. However, the 8-oxodGTPase activity in the liver was 18% (95% CI: 0.2-37%) lower in mice at 25 and 50 weeks than in 10-week-old mice....... The 10- and 100-week-old mice had similar 8-oxodGTPase activity. In contrast, the mRNA expression of Nudt1 was statistically unaltered that likely resulted from higher variation of measurements. The accumulation of 8-oxodG with age is not a direct consequence of decreased enzyme activity toward singlet...

Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

Science.gov (United States)

Bitensky, M.W.; Yoshida, Tatsuro

1997-04-29

A method is disclosed using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4 C storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4 C for prolonged periods of time is achieved by removing oxygen from the red blood cells at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate. 4 figs.

Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+–ATPase Activity and K+/Na+ Homeostasis in Sweet Potato

Directory of Open Access Journals (Sweden)

Yicheng Yu

2018-02-01

Full Text Available Melatonin (MT is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [Ipomoea batatas (L. Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K+/Na+ homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K+/Na+ homeostasis in sweet potato seedlings as indicated by the low reduced K+ content in tissues and low accumulation of Na+ content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K+ efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM H+–ATPase activity and intracellular adenosine triphosphate (ATP level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H+–ATPase activity, and K+/Na+ homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H+–ATPase activity and K+/Na+ homeostasis in sweet potato.

Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4-negative Medium-sized Trigeminal Ganglion Neurons.

Science.gov (United States)

Sato, Masaki; Ogura, Kazuhiro; Kimura, Maki; Nishi, Koichi; Ando, Masayuki; Tazaki, Masakazu; Shibukawa, Yoshiyuki

2018-04-27

Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X 3 ), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B 4 -negative medium-sized neurons. Action potentials in these isolectin B 4 -negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. Odontoblasts established neurotransmission with myelinated Aδ neurons via P2X 3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X 3 receptors induced an action potential

Tools and drugs for uracil nucleotide-activated P2Y receptors.

Science.gov (United States)

Rafehi, Muhammad; Müller, Christa E

2018-04-13

P2Y receptors (P2YRs) are a family of G protein-coupled receptors activated by extracellular nucleotides. Physiological P2YR agonists include purine and pyrimidine nucleoside di- and triphosphates, such as ATP, ADP, UTP, UDP, nucleotide sugars, and dinucleotides. Eight subtypes exist, P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 , which represent current or potential future drug targets. Here we provide a comprehensive overview of ligands for the subgroup of the P2YR family that is activated by uracil nucleotides: P2Y 2 (UTP, also ATP and dinucleotides), P2Y 4 (UTP), P2Y 6 (UDP), and P2Y 14 (UDP, UDP-glucose, UDP-galactose). The physiological agonists are metabolically unstable due to their fast hydrolysis by ectonucleotidases. A number of agonists with increased potency, subtype-selectivity and/or enzymatic stability have been developed in recent years. Useful P2Y 2 R agonists include MRS2698 (6-01, highly selective) and PSB-1114 (6-05, increased metabolic stability). A potent and selective P2Y 2 R antagonist is AR-C118925 (10-01). For studies of the P2Y 4 R, MRS4062 (3-15) may be used as a selective agonist, while PSB-16133 (10-06) represents a selective antagonist. Several potent P2Y 6 R agonists have been developed including 5-methoxyuridine 5'-O-((R p )α-boranodiphosphate) (6-12), PSB-0474 (3-11), and MRS2693 (3-26). The isocyanate MRS2578 (10-08) is used as a selective P2Y 6 R antagonist, although its reactivity and low water-solubility are limiting. With MRS2905 (6-08), a potent and metabolically stable P2Y 14 R agonist is available, while PPTN (10-14) represents a potent and selective P2Y 14 R antagonist. The radioligand [ 3 H]UDP can be used to label P2Y 14 Rs. In addition, several fluorescent probes have been developed. Uracil nucleotide-activated P2YRs show great potential as drug targets, especially in inflammation, cancer, cardiovascular and neurodegenerative diseases. Copyright © 2018. Published by Elsevier Inc.

Dielectric properties of residual water in amorphous lyophilized mixtures of sugar and drug

Energy Technology Data Exchange (ETDEWEB)

Moznine, R El [School of Pharmacy, De Montfort University, Leiceste (United Kingdom); Smith, G [School of Pharmacy, De Montfort University, Leicester (United Kingdom); Polygalov, E [School of Pharmacy, De Montfort University, Leicester (United Kingdom); Suherman, P M [School of Pharmacy, De Montfort University, Leicester (United Kingdom); Broadhead, J [AstraZeneca Charnwood R and D, Bakewell Rd, Loughborough (United Kingdom)

2003-02-21

Dielectric relaxation spectroscopy was used to investigate the properties of residual water in lyophilized formulations of a proprietary tri-phosphate drug containing a sugar (trehalose, lactose or sucrose) or dextran. The dielectric properties of each formulation were determined in the frequency range (0.1 Hz-0.1 MHz) and temperature range (30 deg. C-T{sub g}). The temperature dependence of the relaxation times for all samples showed Arrhenuis behaviour, from which the activation energy was derived. Proton hopping through the hydrogen-bonded network (clusters) of water molecules was suggested as the principle mode of charge transport. Significant differences in dielectric relaxation kinetics and activation energy were observed for the different formulations, which were found to correlate with the amount of monophosphate degradation product.

h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-03-01

1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.

Science.gov (United States)

Kucherenko, I S; Soldatkin, O O; Lagarde, F; Jaffrezic-Renault, N; Dzyadevych, S V; Soldatkin, A P

2015-11-01

Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes. Copyright © 2015 Elsevier B.V. All rights reserved.

h5-HT1B receptor-mediated constitutive Gαi3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-01-01

Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263

Mitochondria, Energy and Cancer: The Relationship with Ascorbic Acid

Science.gov (United States)

González, Michael J.; Rosario-Pérez, Glorivee; Guzmán, Angélica M.; Miranda-Massari, Jorge R.; Duconge, Jorge; Lavergne, Julio; Fernandez, Nadia; Ortiz, Norma; Quintero, Ana; Mikirova, Nina; Riordan, Neil H.; Ricart, Carlos M.

2012-01-01

Ascorbic Acid (AA) has been used in the prevention and treatment of cancer with reported effectiveness. Mitochondria may be one of the principal targets of ascorbate's cellular activity and it may play an important role in the development and progression of cancer. Mitochondria, besides generating adenosine triphosphate (ATP), has a role in apoptosis regulation and in the production of regulatory oxidative species that may be relevant in gene expression. At higher concentrations AA may increase ATP production by increasing mitochondrial electron flux, also may induce apoptotic cell death in tumor cell lines, probably via its pro-oxidant action In contrast, at lower concentrations AA displays antioxidant properties that may prevent the activation of oxidant-induced apoptosis. These concentration dependent activities of ascorbate may explain in part the seemingly contradictory results that have been reported previously. PMID:23565030

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues

International Nuclear Information System (INIS)

Tanner, N.K.; Hanna, M.M.; Abelson, J.

1988-01-01

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs

Hydrolysis of aspartic acid phosphoramidate nucleotides: a comparative quantum chemical study.

Science.gov (United States)

Michielssens, Servaas; Tien Trung, Nguyen; Froeyen, Matheus; Herdewijn, Piet; Tho Nguyen, Minh; Ceulemans, Arnout

2009-09-07

L-Aspartic acid has recently been found to be a good leaving group during HIV reverse transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. This showed that L-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis of several models for L-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic environment was mimicked by involving two Mg(2+) ions and some important active site residues in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier is lower than the comparable mechanism with methanol as a leaving group. The calculations on the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for L-aspartic-dAMP than for L-malic-dAMP and pyrophosphate.

Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells

International Nuclear Information System (INIS)

Morita, T.; Nakamura, H.; Tsutsui, Y.; Nishiyama, Y.; Yoshida, S.

1982-01-01

Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2',3'-dideoxythymidine 5'-triphosphate (d 2 TTP) inhibits DNA polymerase ν and ν but not α. 1-ν-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase α and ν although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-ν-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d 2 Thd, a precursor of d 2 TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d 2 Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d 2 TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d 2 TTP effectively inhibited repair synthesis. The microinjection of d 2 TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis

Nitric Oxide Binds to and Modulates the Activity of a Pollen Specific Arabidopsis Diacylglycerol Kinase

KAUST Repository

Wong, Aloysius Tze

2014-06-01

Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

KAUST Repository

Lee, Seung-Joo

2010-03-28

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

Effect of aging on phosphate metabolites of rat brain as revealed by the in vivo and in vitro 31P NMR measurements

International Nuclear Information System (INIS)

Liu, Hsiuchih; Chi, Chinwen; Liu, Tsungyun; Liu, Lianghui; Luh, Wenming; Hsieh, Changhuain; Wu, Wenguey

1991-01-01

Changes of phosphate metabolism in brains of neonate, weaning and adult rats were compared using both in vivo and in vitro nuclear magnetic resonance spectra. Ratios of phosphocreatine/nucleoside triphosphate (PCr/NTP) were the same in neonatal brain in both in vivo and in vitro studies, but not in weaning and adult brains. This discrepancy may have resulted from extended cerebral hypoxia due to slowed freezing of the brain by the increased skull thickness and brain mass in the weaning and adult rats. Variations of in vitro extraction condition for this age-related study may lead to systematic errors in the adult rats. Nevertheless, the phosphomonoester/nucleoside triphosphate (PME/NTP) ratios in extracts of brain from neonatal rats were higher than those obtained in vivo. In addition, the glycerophosphorylethanolamine plus glycerophosphorylcholine/nucleoside triphosphate (GPE+GPC/NTP) ratios, which were not measurable in vivo, showed age-dependent increase in extracts of rat brain. Some of the phosphomonoester and phosphodiester molecules in rat brain may be undetectable in in vivo NMR analysis because of their interaction with cellular components. The total in vitro GPE and GPC concentration in brain from neonatal rat was estimated to be 0.34 mmole/g wet tissue

Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans

Science.gov (United States)

Stepchenkova, Elena I.; Tarakhovskaya, Elena R.; Spitler, Kathryn; Frahm, Christin; Menezes, Miriam R.; Simone, Peter D.; Kolar, Carol; Marky, Luis A.; Borgstahl, Gloria E. O.; Pavlov, Youri I.

2009-01-01

Sanitization of the cellular nucleotide pools from mutagenic base analogs is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic and toxic incorporation of purine base analogs (i.e. ITP, dITP, XTP, dXTP or 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele 94C->A encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analog drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by western blot is severely diminished in a human fibroblast cell line with the 94C->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability and availability. PMID:19631656

Enhanced antitumor activity of 3-bromopyruvate in combination with rapamycin in vivo and in vitro.

Science.gov (United States)

Zhang, Qi; Pan, Jing; Lubet, Ronald A; Komas, Steven M; Kalyanaraman, Balaraman; Wang, Yian; You, Ming

2015-04-01

3-Bromopyruvate (3-BrPA) is an alkylating agent and a well-known inhibitor of energy metabolism. Rapamycin is an inhibitor of the serine/threonine protein kinase mTOR. Both 3-BrPA and rapamycin show chemopreventive efficacy in mouse models of lung cancer. Aerosol delivery of therapeutic drugs for lung cancer has been reported to be an effective route of delivery with little systemic distribution in humans. In this study, 3-BrPA and rapamycin were evaluated in combination for their preventive effects against lung cancer in mice by aerosol treatment, revealing a synergistic ability as measured by tumor multiplicity and tumor load compared treatment with either single-agent alone. No evidence of liver toxicity was detected by monitoring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. To understand the mechanism in vitro experiments were performed using human non-small cell lung cancer (NSCLC) cell lines. 3-BrPA and rapamycin also synergistically inhibited cell proliferation. Rapamycin alone blocked the mTOR signaling pathway, whereas 3-BrPA did not potentiate this effect. Given the known role of 3-BrPA as an inhibitor of glycolysis, we investigated mitochondrial bioenergetics changes in vitro in 3-BrPA-treated NSCLC cells. 3-BrPA significantly decreased glycolytic activity, which may be due to adenosine triphosphate (ATP) depletion and decreased expression of GAPDH. Our results demonstrate that rapamycin enhanced the antitumor efficacy of 3-BrPA, and that dual inhibition of mTOR signaling and glycolysis may be an effective therapeutic strategy for lung cancer chemoprevention. ©2015 American Association for Cancer Research.

BIOCHEMICAL AND PHYLOGENETIC STUDIES OF CreD OF Corynebacterium glutamicum

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Muhammad Tausif Chaudhry

2015-06-01

Full Text Available CreD characterized as Mg2+-dependent phosphohydrolase with conserved HD domain was involved in 4-cresol metabolism in Corynebacterium glutamicum. Native molecular mass of 54 kDa suggested that the biological unit is a dimer. No deoxynucleotide triphosphate triphosphohydrolase (dNTPase activity was detected for CreD. The apparent Km and Vmax values for 4-nitrophenyl phosphate were 0.35 mM and 16.23 M min-1 mg-1, respectively, while calculated values for kcat and kcat/Km were 0.4 s-1 and 1.14103 M-1 s-1, respectively. Among thiol group inhibitors, iodoacetic acid significantly inhibited phosphohydrolase activity. Sequence identity and phylogenetic analysis suggested universal existence of CreD homologues. Involvement of HD-domain hydrolase in aromatic degradation has not been reported before.

Adaptation of red cell enzymes and intermediates in metabolic disorders.

Science.gov (United States)

Goebel, K M; Goebel, F D; Neitzert, A; Hausmann, L; Schneider, J

1975-01-01

The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.

Peptide YY (3-36) modulates intracellular calcium through activation of the phosphatidylinositol pathway in hippocampal neurons.

Science.gov (United States)

Domingues, Michelle Flores; de Assis, Dênis Reis; Piovesan, Angela Regina; Belo, Cháriston André Dal; da Costa, Jaderson Costa

2018-02-01

Peptide YY (PYY) belongs to the neuropeptide Y (NPY) family, which also includes the pancreatic polypeptide (PP) and NPY. PYY is secreted by the intestinal L cells, being present in the blood stream in two active forms capable of crossing the blood brain barrier, PYY (1-36) and its cleavage product, PYY (3-36). PYY is a selective agonist for the Y2 receptor (Y2R) and these receptors are abundant in the hippocampus. Here we investigated the mechanisms by which PYY (3-36) regulates intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in hippocampal neurons by employing a calcium imaging technique in hippocampal cultures. Alterations in [Ca 2+ ] i were detected by changes in the Fluo-4 AM reagent emission. PYY (3-36) significantly increased [Ca 2+ ] from the concentration of 10 -11 M as compared to the controls (infusion of HEPES-buffered solution (HBS) solution alone). The PYY (3-36)-increase in [Ca 2+ ] i remained unchanged even in Ca 2+ -free extracellular solutions. Sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase pump (SERCA pump) inhibition partially prevent the PYY (3-36)-increase of [Ca 2+ ] i and inositol 1,4,5-triphosphate receptor (IP3R) inhibition also decreased the PYY (3-36)-increase of [Ca 2+ ] i . Taken together, our data strongly suggest that PYY (3-36) mobilizes calcium from the neuronal endoplasmic reticulum (ER) stores towards the cytoplasm. Next, we showed that PYY (3-36) inhibited high K + -induced increases of [Ca 2+ ] i , suggesting that PYY (3-36) could also act by activating G-protein coupled inwardly rectifying potassium K + channels. Finally, the co-infusion of the Y2 receptor (Y2R) antagonist BIIE0246 with PYY (3-36) abolished the [Ca 2+ ] i increase induced by the peptide, suggesting that PYY (3-36)-induced [Ca 2+ ] i increase in hippocampal neurons occurs via Y2Rs. Copyright © 2017 Elsevier Ltd. All rights reserved.

/sup 1/H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla

Energy Technology Data Exchange (ETDEWEB)

MacD. Stewart, J.M.; Grisham, C.M.

1988-06-28

/sup 1/H nuclear magnetic relaxation measurements have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH/sub 3/)/sub 4/ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH/sub 4/)/sub 4/ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase and that Mn/sup 2 +/ bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na-K-ATPase. From the paramagnetic effect of Mn/sup 2 +/ bound to the APTase on the longitudinal relaxation rates of the protons of Co(NH/sub 3/)/sub 4/ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous /sup 31/P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35/sup 0/, and the conformation of the ribose ring is slightly N-type. The bound Mn/sup 2 +/ lies above and in the plane of the adenine ring. The distances from Mn/sup 2 +/ to N/sub 6/ and N/sub 7/ are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

Science.gov (United States)

Morin, Benjamin; Liang, Bo; Gardner, Erica; Ross, Robin A; Whelan, Sean P J

2017-01-01

We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L

Comparative enzymology of the adenosine triphosphate sulfurylases from leaf tissue of selenium-accumulator and non-accumulator plants

Energy Technology Data Exchange (ETDEWEB)

Shaw, W H; Anderson, J W

1974-01-01

ATP sulfurylases were partially purified (20-40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulfate-dependent PP/sub 1/-ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulfurylases were similar to the spinach enzyme. The ATP sulfurylases from both selenium-accumulator and non-accumulator species catalyzed selenate-dependent PP/sub 1/-ATP exchange; selenate competed with sulfate. The ratio of V(selenate)/V(sulfate) and K/sub m/ (selenate)/K/sub m/(sulfate) was approximately the same for the enzyme from each species. Sulfate-dependent PP/sub 1/-ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanisms, in which ATP is the first substrate to react with the enzyme and PP/sub 1/ is the first product released. Synthesis of adenosine 5'-(/sup 35/S)sulfatophosphate from (/sup 35/S)sulfate was demonstrated by coupling the Astrgalus ATP sulfurylases with Mg/sup 2 +/-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using (/sup 75/Se)selenate as substrate could not be demonstrated.

Prebiotic Factors Influencing the Activity of a Ligase Ribozyme

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Fabrizio Anella

2017-04-01

Full Text Available An RNA-lipid origin of life scenario provides a plausible route for compartmentalized replication of an informational polymer and subsequent division of the container. However, a full narrative to form such RNA protocells implies that catalytic RNA molecules, called ribozymes, can operate in the presence of self-assembled vesicles composed of prebiotically relevant constituents, such as fatty acids. Hereby, we subjected a newly engineered truncated variant of the L1 ligase ribozyme, named tL1, to various environmental conditions that may have prevailed on the early Earth with the objective to find a set of control parameters enabling both tL1-catalyzed ligation and formation of stable myristoleic acid (MA vesicles. The separate and concurrent effects of temperature, concentrations of Mg2+, MA, polyethylene glycol and various solutes were investigated. The most favorable condition tested consists of 100 mM NaCl, 1 mM Mg2+, 5 mM MA, and 4 °C temperature, whereas the addition of Mg2+-chelating solutes, such as citrate, tRNAs, aspartic acid, and nucleoside triphosphates severely inhibits the reaction. These results further solidify the RNA-lipid world hypothesis and stress the importance of using a systems chemistry approach whereby a wide range of prebiotic factors interfacing with ribozymes are considered.

A Role for the Krebs Cycle Intermediate Citrate in Metabolic Reprogramming in Innate Immunity and Inflammation

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Niamh C. Williams

2018-02-01

Full Text Available Metabolism in immune cells is no longer thought of as merely a process for adenosine triphosphate (ATP production, biosynthesis, and catabolism. The reprogramming of metabolic pathways upon activation is also for the production of metabolites that can act as immune signaling molecules. Activated dendritic cells (DCs and macrophages have an altered Krebs cycle, one consequence of which is the accumulation of both citrate and succinate. Citrate is exported from the mitochondria via the mitochondrial citrate- carrier. Cytosolic metabolism of citrate to acetyl-coenzyme A (acetyl-CoA is important for both fatty-acid synthesis and protein acetylation, both of which have been linked to macrophage and DC activation. Citrate-derived itaconate has a direct antibacterial effect and also has been shown to act as an anti-inflammatory agent, inhibiting succinate dehydrogenase. These findings identify citrate as an important metabolite for macrophage and DC effector function.

Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

Science.gov (United States)

Figueroa, Vania A.; Retamal, Mauricio A.; Cea, Luis A.; Salas, José D.; Vargas, Aníbal A.; Verdugo, Christian A.; Jara, Oscar; Martínez, Agustín D.; Sáez, Juan C.

2014-01-01

Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects. PMID:25237294

Integrated approach to characterize fouling on a flat sheet membrane gravity driven submerged membrane bioreactor

KAUST Repository

Fortunato, Luca; Jeong, Sanghyun; Wang, Yiran; Behzad, Ali Reza; Leiknes, TorOve

2016-01-01

of different analytical tools, including optical coherence tomography (OCT), liquid chromatography with organic carbon detection (LC-OCD), total organic carbon (TOC), flow cytometer (FCM), adenosine triphosphate analysis (ATP) and scanning electron microscopy

Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release.

Science.gov (United States)

Cook, Zara C; Gray, Michael A; Cann, Martin J

2012-07-27

Elevated CO(2) is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO(2) blunts G protein-activated cAMP signaling. The effect of CO(2) is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO(2) on cAMP levels required the activity of the IP(3) receptor. Consistent with these findings, CO(2) caused an increase in steady state cytoplasmic Ca(2+) concentrations not observed in the absence of the IP(3) receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na(+)/H(+) antiporter (NHE3) to demonstrate a functional relevance for CO(2)-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO(2) abrogated the inhibitory effect of cAMP on NHE3 function via an IP(3) receptor-dependent mechanism.

Elevated Carbon Dioxide Blunts Mammalian cAMP Signaling Dependent on Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Release*

Science.gov (United States)

Cook, Zara C.; Gray, Michael A.; Cann, Martin J.

2012-01-01

Elevated CO2 is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO2 blunts G protein-activated cAMP signaling. The effect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO2 on cAMP levels required the activity of the IP3 receptor. Consistent with these findings, CO2 caused an increase in steady state cytoplasmic Ca2+ concentrations not observed in the absence of the IP3 receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na+/H+ antiporter (NHE3) to demonstrate a functional relevance for CO2-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO2 abrogated the inhibitory effect of cAMP on NHE3 function via an IP3 receptor-dependent mechanism. PMID:22654111

Self-Delivery Nanoparticles of Amphiphilic Methotrexate-Gemcitabine Prodrug for Synergistic Combination Chemotherapy via Effect of Deoxyribonucleotide Pools.

Science.gov (United States)

Wang, Yao; Huang, Ping; Hu, Minxi; Huang, Wei; Zhu, Xinyuan; Yan, Deyue

2016-11-16

The distinct and complementary biochemical mechanisms of folic acid analog methotrexate (MTX) and cytidine analog gemcitabine (GEM) make their synergistic combination effective. Unfortunately, such a combination faces severe pharmacokinetic problems and several transportation barriers. To overcome these problems, a new strategy of amphiphilic small molecule prodrug (ASMP) is developed to improve their synergistic combination effect. The ASMP was prepared by the amidation of the hydrophilic GEM with the hydrophobic MTX at a fixed ratio. Owing to its inherent amphiphilicity, the MTX-GEM ASMP self-assembled into stable nanoparticles (ASMP-NPs) with high drug loading capacity (100%), in which the MTX and GEM could self-deliver without any carriers and release synchronously in cancer cells. In vitro studies showed that the MTX-GEM ASMP-NPs could greatly improve the synergistic combination effects by the reason of arresting more S phase of the cell cycle and reducing levels of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), and deoxycytidine triphosphate (dCTP). The stronger synergistic effects caused the higher cell cytotoxicity and apoptotic ratio, and circumvented the multidrug resistance (MDR) of tumor cells. Additionally, MTX-GEM ASMP-NPs could achieve the same anticancer effect with the greatly reduced dosage compared with the free drugs according to the dose-reduction index (DRI) values of MTX and GEM in MTX-GEM ASMP-NPs, which may be beneficial for reducing the side effects.

Long-term performance and fouling analysis of full-scale direct nanofiltration (NF) installations treating anoxic groundwater

KAUST Repository

Beyer, Florian; Rietman, Bas M.; Zwijnenburg, Arie; Van Den Brink, Paula J.; Vrouwenvelder, Johannes S.; Jarzembowska, Monika; Laurinonyte, Judita; Stams, Alfons JM M; Plugge, Caroline M.

2014-01-01

. Investigations using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), total organic carbon (TOC) and adenosine triphosphate (ATP) measurements revealed a complex mixture of organic, biological and inorganic materials. The fouling

[Thiamine and its derivatives in the regulation of cell metabolism].

Science.gov (United States)

Tylicki, Adam; Siemieniuk, Magdalena

2011-07-06

For over 70 years thiamine (vitamin B1) has aroused the interest of biologists, biochemists and medical doctors because of its multilateral participation in key biochemical and physiological processes. The thiamine molecule is composed of pyrimidine and thiazole rings which are linked by a methylene bridge. It is synthesized by microorganisms, fungi and plants, whereas animals and humans have to obtain it from food. There are several known forms of vitamin B1 inside cells: free thiamine, three phosphate esters (mono-, di-, and triphosphate), and the recently found adenosine thiamine triphosphate. Thiamine has a dual, coenzymatic and non-coenzymatic role. First of all, it is a precursor of thiamin diphosphate, which is a coenzyme for over 20 characterized enzymes which are involved in cell bioenergetic processes leading to the synthesis of ATP. Moreover, these enzymes take part in the biosynthesis of pentose (required for the synthesis of nucleotides), amino acids and other organic compounds of cell metabolism. On the other hand, recent discoveries show the non-coenzymatic role of thiamine derivatives in the process of regulation of gene expression (riboswitches in microorganisms and plants), the stress response, and perhaps so far unknown signal transduction pathways associated with adverse environmental conditions, or transduction of nerve signals with participation of thiamine triphosphate and adenosine thiamine triphosphate. From the clinical point of view thiamine deficiency is related to beri-beri, Parkinson disease, Alzheimer disease, Wernicke-Korsakoff syndrome and other pathologies of the nervous system, and it is successfully applied in medical practice. On the other hand, identifying new synthetic analogues of thiamine which could be used as cytostatics, herbicides or agents preventing deficiency of vitamin B1 is currently the major goal of the research. In this paper we present the current state of knowledge of thiamine and its derivatives, indicating

Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

Science.gov (United States)

Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

2014-09-05

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

International Nuclear Information System (INIS)

Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

1997-01-01

Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

Eukaryotic inhibitors or activators elicit responses to chemosensory compounds by ruminal isotrichid and entodiniomorphid protozoa.

Science.gov (United States)

Diaz, H L; Barr, K N; Godden, K R; Plank, J E; Zapata, I; Schappacher, A N; Wick, M P; Firkins, J L

2014-01-01

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the

You Are What You Eat

Science.gov (United States)

... by generating adenosine triphosphate, or ATP, the main currency of metabolism. In addition to providing and storing ... wafer-like chips similar in size to those used in computers. Amino acids link head-to-tail ...

Dietary strategies to treat hyperhomocysteinaemia based on the ...

African Journals Online (AJOL)

2014-01-13

Jan 13, 2014 ... of a methyl group and the purine base, adenine (from adenosine triphosphate or ..... rat liver betaine‑homocysteine methyltransferase gene expression and organization of the .... Betaine rescue of an animal model with.

The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization.

Directory of Open Access Journals (Sweden)

Tobias Maisel

Full Text Available CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO₂] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk, preparations of CoxD revealed a mean K(M value of 0.69±0.14 mM ATP and an apparent V(max value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer, preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO₂] cluster assembly are discussed.

Limonene protects osteoblasts against methylglyoxal-derived adduct formation by regulating glyoxalase, oxidative stress, and mitochondrial function.

Science.gov (United States)

Suh, Kwang Sik; Chon, Suk; Choi, Eun Mi

2017-12-25

Methylglyoxal (MG) is a potent protein glycating agent and an important precursor of advanced glycation end products, which are involved in the pathogenesis of diabetic osteopathy. In this study, we investigated the effects of limonene on MG-induced damage in osteoblastic MC3T3-E1 cells. Pretreating cells with limonene prevented MG-induced protein adduct formation, tumor necrosis factor alpha and interleukin-6 release, mitochondrial superoxide production, and cardiolipin peroxidation. In addition, limonene increased glyoxalase I activity, and glutathione and heme oxygenase-1 levels in the presence of MG. Pretreatment with limonene prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss, and reduced the levels of adenosine monophosphate-activated protein kinase, peroxisome proliferator activated receptor γ coactivator 1α, and nitric oxide. These results demonstrate that limonene may prevent the development of diabetic osteopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

Sleep and immune function: glial contributions and consequences of aging.

Science.gov (United States)

Ingiosi, Ashley M; Opp, Mark R; Krueger, James M

2013-10-01

The reciprocal interactions between sleep and immune function are well-studied. Insufficient sleep induces innate immune responses as evidenced by increased expression of pro-inflammatory mediators in the brain and periphery. Conversely, immune challenges upregulate immunomodulator expression, which alters central nervous system-mediated processes and behaviors, including sleep. Recent studies indicate that glial cells, namely microglia and astrocytes, are active contributors to sleep and immune system interactions. Evidence suggests glial regulation of these interactions is mediated, in part, by adenosine and adenosine 5'-triphosphate actions at purinergic type 1 and type 2 receptors. Furthermore, microglia and astrocytes may modulate declines in sleep-wake behavior and immunity observed in aging. Copyright © 2013. Published by Elsevier Ltd.

Sympathetic vasoconstriction takes an unexpected pannexin detour

DEFF Research Database (Denmark)

Schak Nielsen, Morten

2015-01-01

Sympathetic vasoconstriction plays an important role in the control of blood pressure and the distribution of blood flow. In this issue of Science Signaling, Billaud et al. show that sympathetic vasoconstriction occurs through a complex scheme involving the activation of large-pore pannexin 1...... channels and the subsequent release of adenosine triphosphate that promotes contraction in an autocrine and paracrine manner. This elaborate mechanism may function as a point of intercept for other signaling pathways-for example, in relation to the phenomenon "functional sympatholysis," in which exercise...... abrogates sympathetic vasoconstriction in skeletal muscle. Because pannexin 1 channels are inhibited by nitric oxide, they may function as a switch to turn off adrenergic signaling in skeletal muscle during exercise....

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.

Science.gov (United States)

Sarkar, Sailendra Nath; Bakshi, Sankar; Mokkapati, Sanath K; Roy, Sujit; Sengupta, Dibyendu N

2004-07-16

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.

UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli

International Nuclear Information System (INIS)

Das, S.K.; Loeb, L.A.

1984-01-01

UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP. These selective increases in dATP and dTTP pools are seen in wild-type E. coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose. The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed. (orig.)

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

KAUST Repository

Wong, Aloysius Tze; Gehring, Christoph A

2013-01-01

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

Sequence Classification: 203907 [

Lifescience Database Archive (English)

Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|71891851|ref|YP_277580.1| dihydrone...opterin aldolase, also has dihydroneopterin triphosphate || http://www.ncbi.nlm.nih.gov/protein/71891851 ...

Studies towards the synthesis of ATP analogs as potential glutamine synthetase inhibitors

CSIR Research Space (South Africa)

Salisu, S

2011-05-01

Full Text Available and Centre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown, South Africa b CSIR BIO/CHEMTEK, Modderfontein, South Africa ABSTRACT In research directed at the development of adenine triphosphate (ATP) analogs as potential...

Effect of Iboga alkaloids on µ-opioid receptor-coupled G protein activation.

Directory of Open Access Journals (Sweden)

Tamara Antonio

Full Text Available The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of μ-opioid receptor (MOR-related G proteins by iboga alkaloids.Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC, a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio-triphosphate ([(35S]GTPγS binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices.In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine to 13 uM (noribogaine and 18MC. Noribogaine and 18-MC did not stimulate [(35S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [(35S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [(35S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

DEFF Research Database (Denmark)

Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

2010-01-01

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

Effects of ketamine and its isomers on ischemic preconditioning in the isolated rat heart

NARCIS (Netherlands)

Molojavyi, A.; Preckel, B.; Comfère, T.; Müllenheim, J.; Thämer, V.; Schlack, W.

2001-01-01

BACKGROUND: Ischemic preconditioning protects the heart against subsequent ischemia. Opening of the adenosine triphosphate-sensitive potassium (KATP) channel is a key mechanism of preconditioning. Ketamine blocks KATP channels of isolated cardiomyocytes. The authors investigated the effects of

Identification and Characterization of Novel Plant Adenylate Cyclases – The Arabidopsis Thaliana Potassium Uptake Permeases

KAUST Repository

Al-Younis, Inas

2018-01-01

Adenylyl Cyclases (ACs) catalyze the formation of the key universal second messenger adenosine 3’, 5’-cyclic monophosphate (cAMP) from adenosine 5’- triphosphate. Cyclic AMP participates in several signal transduction pathways and is present

The generation of radiolabeled DNA and RNA probes with polymerase chain reaction

International Nuclear Information System (INIS)

Schowalter, D.B.; Sommer, S.S.

1989-01-01

By including a radioactive triphosphate during polymerase chain reaction (PCR), probes of very high specific activity can be generated. The advantages of PCR labeling include (1) uniform labeling with a specific activity of 5 X 10(9) cpm/micrograms or higher (sensitivity of detection: 0.028 pg of target DNA per 24 h); (2) ease of regulation of both the specific activity and the amount of labeled probe produced; (3) efficient labeling of fragments less than 500 bp; (4) efficient incorporation over a wide range of input DNA template; (5) labeling with subnanogram amounts of input DNA; and (6) direct labeling of genomic DNA. The minimal amount of input DNA allows a virtually unlimited number of PCR labeling reactions to be performed on DNA generated by one amplification under the previously described nonlabeling conditions. This obviates the need for CsCl gradients or other large scale methods of DNA preparation. The above advantages except for the very high specific activity can also be achieved by transcript labeling after an amplification where one or both of PCR primers contain a phage promoter sequence

Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom.

Science.gov (United States)

Saoud, Samah; Chérifi, Fatah; Benhassine, Traki; Laraba-Djebari, Fatima

2017-05-01

The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni 2+ and Mg 2+ completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy. © 2016 Wiley Periodicals, Inc.

Fenofibrate plus Metformin Produces Cardioprotection in a Type 2 Diabetes and Acute Myocardial Infarction Model

Directory of Open Access Journals (Sweden)

Víctor Hugo Oidor-Chan

2016-01-01

Full Text Available We investigated whether fenofibrate, metformin, and their combination generate cardioprotection in a rat model of type 2 diabetes (T2D and acute myocardial infarction (AMI. Streptozotocin-induced diabetic- (DB- rats received 14 days of either vehicle, fenofibrate, metformin, or their combination and immediately after underwent myocardial ischemia/reperfusion (I/R. Fenofibrate plus metformin generated cardioprotection in a DBI/R model, reported as decreased coronary vascular resistance, compared to DBI/R-Vehicle, smaller infarct size, and increased cardiac work. The subchronic treatment with fenofibrate plus metformin increased, compared with DBI/R-Vehicle, total antioxidant capacity, manganese-dependent superoxide dismutase activity (MnSOD, guanosine triphosphate cyclohydrolase I (GTPCH-I expression, tetrahydrobiopterin : dihydrobiopterin (BH4 : BH2 ratio, endothelial nitric oxide synthase (eNOS activity, nitric oxide (NO bioavailability, and decreased inducible NOS (iNOS activity. These findings suggest that PPARα activation by fenofibrate + metformin, at low doses, generates cardioprotection in a rat model of T2D and AMI and may represent a novel treatment strategy to limit I/R injury in patients with T2D.

Redox-sensitive alteration of replisome architecture safeguards genome integrity

DEFF Research Database (Denmark)

Somyajit, Kumar; Gupta, Rajat; Sedlackova, Hana

2017-01-01

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX...

Red blood cell phosphate concentration and osmotic resistance during dietary phosphate depletion in dairy cows

NARCIS (Netherlands)

Grünberg, W; Mol, J A; Teske, E

BACKGROUND: Hypophosphatemia in early lactating dairy cows has been implicated as primary cause for postparturient hemoglobinuria in cattle. Decreased availability of phosphorus has been proposed to reduce adenosine triphosphate synthesis of erythrocytes and thereby reduce osmotic resistance of

Rapid detection of E. Coli O157:H7 by IFAST and ATP bioluminescence assay for water analysis

CSIR Research Space (South Africa)

Ngamsom, B

2016-10-01

Full Text Available The present investigation reports isolation and detection of E. coli O157:H7 employing a simple and portable microfluidic device based on immiscible filtration assisted by surface tension (IFAST) and adenosine triphosphate (ATP) bioluminescence...

Kinetics and mechanism of DNA repair

International Nuclear Information System (INIS)

Meldrum, R.A.; Wharton, C.W.; Shall, S.

1990-01-01

Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the γ-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside trisphosphate is isotopically labelled in the α-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair. (author)

Kinetics and mechanism of DNA repair; Evaluation of caged compounds for use in studies of u. v. -induced DNA repair

Energy Technology Data Exchange (ETDEWEB)

Meldrum, R.A.; Wharton, C.W. (Birmingham Univ. (UK). Dept. of Biochemistry); Shall, S. (Sussex Univ., Brighton (UK). School of Biological Sciences)

1990-03-15

Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the {gamma}-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside trisphosphate is isotopically labelled in the {alpha}-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair. (author).

Deprotonated imidodiphosphate in AMPPNP-containing protein structures

International Nuclear Information System (INIS)

Dauter, Miroslawa; Dauter, Zbigniew

2011-01-01

In certain AMPPNP-containing protein structures, the nitrogen bridging the two terminal phosphate groups can be deprotonated. Many different proteins utilize the chemical energy provided by the cofactor adenosine triphosphate (ATP) for their proper function. A number of structures in the Protein Data Bank (PDB) contain adenosine 5′-(β,γ-imido)triphosphate (AMPPNP), a nonhydrolysable analog of ATP in which the bridging O atom between the two terminal phosphate groups is substituted by the imido function. Under mild conditions imides do not have acidic properties and thus the imide nitrogen should be protonated. However, an analysis of protein structures containing AMPPNP reveals that the imide group is deprotonated in certain complexes if the negative charges of the phosphate moieties in AMPPNP are in part neutralized by coordinating divalent metals or a guanidinium group of an arginine

The Shigella flexneri OmpA amino acid residues 188EVQ190 are essential for the interaction with the virulence factor PhoN2.

Science.gov (United States)

Scribano, Daniela; Damico, Rosanna; Ambrosi, Cecilia; Superti, Fabiana; Marazzato, Massimiliano; Conte, Maria Pia; Longhi, Catia; Palamara, Anna Teresa; Zagaglia, Carlo; Nicoletti, Mauro

2016-12-01

Shigella flexneri is an intracellular pathogen that deploys an arsenal of virulence factors promoting host cell invasion, intracellular multiplication and intra- and inter-cellular dissemination. We have previously reported that the interaction between apyrase (PhoN2), a periplasmic ATP-diphosphohydrolase, and the C-terminal domain of the outer membrane (OM) protein OmpA is likely required for proper IcsA exposition at the old bacterial pole and thus for full virulence expression of Shigella flexneri (Scribano et al., 2014). OmpA, that is the major OM protein of Gram-negative bacteria, is a multifaceted protein that plays many different roles both in the OM structural integrity and in the virulence of several pathogens. Here, by using yeast two-hybrid technology and by constructing an in silico 3D model of OmpA from S. flexneri 5a strain M90T, we observed that the OmpA residues 188 EVQ 190 are likely essential for PhoN2-OmpA interaction. The 188 EVQ 190 amino acids are located within a flexible region of the OmpA protein that could represent a scaffold for protein-protein interaction.

Progranulin Reduced Neuronal Cell Death by Activation of Sortilin 1 Signaling Pathways After Subarachnoid Hemorrhage in Rats.

Science.gov (United States)

Li, Bo; He, Yue; Xu, Liang; Hu, Qin; Tang, Junjia; Chen, Yujie; Tang, Jiping; Feng, Hua; Zhang, John H

2015-08-01

Progranulin has been reported to have neuroprotective actions in cultured neurons. This study investigated the effect of recombinant rat progranulin on early brain injury after subarachnoid hemorrhage. Controlled in vivo laboratory study. Animal research laboratory. Two hundred thirty adult male Sprague-Dawley rats weighing 280-320 g. Subarachnoid hemorrhage was induced in rats by endovascular perforation. Rat recombinant progranulin (1 and 3 ng) was administrated intracerebroventricularly at 1.5 hours after subarachnoid hemorrhage. Progranulin small interfering RNA was administrated by intracerebroventricularly at 1 day before subarachnoid hemorrhage induction. Subarachnoid hemorrhage grade, neurologic score, and brain water content were measured at 24 and 72 hours after subarachnoid hemorrhage. Neural apoptosis was evaluated by double immunofluorescence staining using terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick-end labeling and neuronal nuclei. For mechanistic study, the expression of progranulin, phosphorylated Akt, Akt, p-Erk, Erk, Bcl-2, and cleaved caspase-3 were analyzed by Western blot at 24 hours after subarachnoid hemorrhage. siRNA for sortilin 1 (a progranulin receptor) was used to intervene the downstream pathway. The expression of progranulin decreased and reached the lowest point at 24 hours after subarachnoid hemorrhage. Administration of rat recombinant progranulin decreased brain water content and improved neurologic functions at both 24 and 72 hours after subarachnoid hemorrhage, while knockdown of endogenous progranulin aggravated neurologic deficits after subarachnoid hemorrhage. Rat recombinant progranulin treatment reduced neuronal apoptosis, while progranulin deficiency promoted neuronal apoptosis at 24 hours after subarachnoid hemorrhage. Rat recombinant progranulin promoted Akt activation, increased Bcl-2 level, but reduced caspase-3 level. Knockdown of progranulin binding factor sortilin 1

The diagnostic value of immunohistochemically detected methylthioadenosine phosphorylase deficiency in malignant pleural mesotheliomas

DEFF Research Database (Denmark)

Zimling, Zarah Glad; Jørgensen, Anne; Santoni-Rugiu, Eric

2012-01-01

  Malignant pleural mesothelioma (MPM) often causes diagnostic difficulties for pathologists. We assessed whether loss of methylthioadenosine phosphorylase (MTAP), a key enzyme in the intracellular recycling of adenosine triphosphate (ATP) often deleted in MPM, could be detected with immunohistoc...

Homogeneous electrochemical aptamer-based ATP assay with signal amplification by exonuclease III assisted target recycling.

Science.gov (United States)

Liu, Shufeng; Wang, Ying; Zhang, Chengxin; Lin, Ying; Li, Feng

2013-03-21

A novel and homogeneous electrochemical aptamer-based adenosine triphosphate (ATP) assay was demonstrated with signal amplification by exonuclease III-assisted target recycling. A superior detection limit of 1 nM toward ATP with an excellent selectivity could be achieved.

Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches

NARCIS (Netherlands)

Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.W.G.; Haastert, van P.J.M.

2004-01-01

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

NARCIS (Netherlands)

Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.; van Haastert, P.J.

2004-01-01

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate