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Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation.  


In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk. PMID:18726586

Ishida, Akihiko; Yamada, Yasuko; Kamidate, Tamio



Inhibition of the Fe(III)-catalyzed dopamine oxidation by ATP and its relevance to oxidative stress in Parkinson's disease.  


Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity. PMID:23823941

Jiang, Dianlu; Shi, Shuyun; Zhang, Lin; Liu, Lin; Ding, Bingrong; Zhao, Bingqing; Yagnik, Gargey; Zhou, Feimeng



Mitochondrial complex III defects contribute to inefficient respiration and ATP synthesis in the myocardium of Trypanosoma cruzi-infected mice.  


In this study, we conducted a thorough analysis of mitochondrial bioenergetic function as well as the biochemical and molecular factors that are deregulated and contribute to compromised adenosine triphosphate (ATP) production in the myocardium during Trypanosoma cruzi infection. We show that ADP-stimulated state 3 respiration and ATP synthesis supported by pyruvate/malate (provides electrons to complex I) and succinate (provides electrons to complex II) substrates were significantly decreased in left ventricular tissue and isolated cardiac mitochondria of infected mice. The decreased mitochondrial ATP synthesis in infected murine hearts was not a result of uncoupling between the electron-transport chain and oxidative phosphorylation and decreased availability of the intermediary metabolites (e.g., NADH). The observed decline in the activities of complex-I, -IV, and -V was not physiologically relevant and did not contribute to compromised respiration and ATP synthesis in infected myocardium. Instead, complex III activity was decreased above the threshold level and contributed to respiratory-chain inefficiency and the resulting decline in mitochondrial ATP synthesis in infected myocardium. The loss in complex III activity occurred as a consequence of cytochrome b depletion. Treatment of infected mice with phenyl-alpha-tert-butyl nitrone (PBN, antioxidant) was beneficial in preserving the mtDNA-encoded cytochrome b expression, and subsequently resulted in improved complex III activity, mitochondrial respiration, and ATP production in infected myocardium. Overall, we provide novel data on the mechanism(s) involved in cardiac bioenergetic inefficiency during T. cruzi infection. PMID:19624257

Wen, Jian-Jun; Garg, Nisha Jain



Metabolic syndrome prevalence according to ATP III and IDF criteria and related factors in Turkish adults  

PubMed Central

Introduction The aim of this study is to investigate the prevalence of metabolic syndrome (MS) and its components according to Adult Treatment Panel III (ATP III) and International Diabetes Federation (IDF) criteria and the risk factors affecting MS. Metabolic syndrome prevalence was evaluated according to certain quintet age groups, altitude, location and demographic features. Material and methods This study was a cross-sectional survey conducted in 24 provinces from the 7 regions of Turkey. A total of 4309 adults from 7 regions participated in the study (1947 males, 45.2%). Results The mean age of participants was 47 ±14 years. Metabolic syndrome prevalence was found as 36.6% according to ATP III and 44.0% according to IDF. The MS rate was found to be higher in females compared to males in both groups (p < 0.01). According to both criteria, MS prevalence was found to be higher in subjects who lived in coastal regions when evaluated according to altitude and in subjects who lived in district centers when evaluated according to location. The MS risk is 1.62-fold higher in females compared to males. Metabolic syndrome risk increases as age increases and is highest in the 61-65 age group. Metabolic syndrome risk increases 2.75-fold in the overweight compared to normal weighing subjects and 7.80-fold in the obese. Conclusions Metabolic syndrome prevalence was found to be high in Turkey according to both criteria. Metabolic syndrome prevalence increases as age and body mass index (BMI) increase. Age, female gender and obesity are independent risk factors for MS development.

Bayram, Fahri; Gedik, Vedia; Kaya, Ahmet; Karaman, Ahmet; Demir, Ozgur; Sabuncu, Tevfik; Kocer, Derya; Coskun, Ramazan



Disorder as Harmful Dysfunction: A Conceptual Critique of "DSM-III-R's" Definition of Mental Disorder.  

ERIC Educational Resources Information Center

The definition of mental disorder in the third edition of the "Diagnostic and Statistical Manual of Mental Disorders" (DSM-III-R) explains that a disorder is harmful and represents a dysfunction, but it fails to capture the idea of dysfunction. Ideas for increasing the conceptual validity of the definition are presented. (SLD)

Wakefield, Jerome C.



The prevalence of metabolic syndrome according to the Iranian Committee of Obesity and ATP III criteria in Babol, North of Iran.  

PubMed Central

Background: Metabolic syndrome (MS) is highly significant due to its association to type 2 diabetes and cardiovascular diseases. The purpose of this study was to compare the prevalence of MS according to the report of the Iranian National Committee of Obesity criteria (INCO) versus Adult Treatment Panel III (ATPIII) in Babol, North of Iran. Methods: Data obtained based on criteria ATP III from the Babol Lipid and Glucose Study (from July 2004 to September 2005) and were compared with the new INCO criteria 2010. The data were collected and analyzed. Results: In total, 933 adult males and females were evaluated. According to ATP III criteria, the overall prevalence of metabolic syndrome was 23.7% (95% confidence interval: 21%-26.4%); 28.4% and 9.4% were females and males, respectively; however, the prevalence was 20.5% (95% confidence interval: 17.9%?23.1%) according to the INCO criteria, 22.5% and 15.7% were females and males, respectively. Conclusion: The new INCO criteria for the metabolic syndrome proclaimed by the Iranian Committee of Obesity estimated a lower prevalence of syndrome in comparison with ATP III criteria in Babol.

Mahjoub, Soleiman; Haji Ahmadi, Mahmoud; Faramarzi, Mahbobeh; Ghorbani, Hiva; Moazezi, Zoleika



The Association Between the Receipt of Lipid Lowering Therapy and HIV Status Among Veterans Who Met NCEP\\/ATP III Criteria for the Receipt of Lipid Lowering Medication  

Microsoft Academic Search

OBJECTIVE  To examine the association between HIV infection status and the receipt of lipid lowering therapy based on National Cholesterol\\u000a Education Program\\/Adult Treatment Panel (NCEP\\/ATP III) guidelines and to assess whether HIV viral load and hepatitis C (HCV)\\u000a status alters that association.\\u000a \\u000a \\u000a \\u000a PARTICIPANTS AND DESIGN  A cross-sectional analysis of survey, laboratory, and pharmacy data from 1,577 male participants (59% HIV infected) of

Matthew S. Freiberg; David A. Leaf; Joseph L. Goulet; Matthew B. Goetz; Krisann K. Oursler; Cynthia L. Gibert; Maria C. Rodriguez-Barradas; Adeel A. Butt; Amy C. Justice



In vitro study of accuracy of cervical pedicle screw insertion using an electronic conductivity device (ATPS part III)  

PubMed Central

Reconstruction of the highly unstable, anteriorly decompressed cervical spine poses biomechanical challenges to current stabilization strategies, including circumferential instrumented fusion, to prevent failure. To avoid secondary posterior surgery, particularly in the elderly population, while increasing primary construct rigidity of anterior-only reconstructions, the authors introduced the concept of anterior transpedicular screw (ATPS) fixation and plating. We demonstrated its morphological feasibility, its superior biomechanical pull-out characteristics compared with vertebral body screws and the accuracy of inserting ATPS using a manual fluoroscopically assisted technique. Although accuracy was high, showing non-critical breaches in the axial and sagittal plane in 78 and 96%, further research was indicated refining technique and increasing accuracy. In light of first clinical case series, the authors analyzed the impact of using an electronic conductivity device (ECD, PediGuard) on the accuracy of ATPS insertion. As there exist only experiences in thoracolumbar surgery the versatility of the ECD was also assessed for posterior cervical pedicle screw fixation (pCPS). 30 ATPS and 30 pCPS were inserted alternately into the C3–T1 vertebra of five fresh-frozen specimen. Fluoroscopic assistance was only used for the entry point selection, pedicle tract preparation was done using the ECD. Preoperative CT scans were assessed for sclerosis at the pedicle entrance or core, and vertebrae with dense pedicles were excluded. Pre- and postoperative reconstructed CT scans were analyzed for pedicle screw positions according to a previously established grading system. Statistical analysis revealed an astonishingly high accuracy for the ATPS group with no critical screw position (0%) in axial or sagittal plane. In the pCPS group, 88.9% of screws inserted showed non-critical screw position, while 11.1% showed critical pedicle perforations. The usage of an ECD for posterior and anterior pedicle screw tract preparation with the exclusion of dense cortical pedicles was shown to be a successful and clinically sound concept with high-accuracy rates for ATPS and pCPS. In concert with fluoroscopic guidance and pedicle axis views, application of an ECD and exclusion of dense cortical pedicles might increase comfort and safety with the clinical use of pCPS. In addition, we presented a reasonable laboratory setting for the clinical introduction of an ATPS-plate system.

Hitzl, Wolfgang; Acosta, Frank; Tauber, Mark; Zenner, Juliane; Resch, Herbert; Yukawa, Yasutsugu; Meier, Oliver; Schmidt, Rene; Mayer, Michael



Calcium binding to the chloroplast and E. coli (CF 0 ) F 0 subunit III (c) of the ATP-synthase  

Microsoft Academic Search

Subunit III and c, the 8 kDa components of the chloroplast CF0, andE. coli H+ channel complexes respectively, were isolated and purified for the purpose of studying their Ca++-binding properties. Purified subunit III or c as well as the unfractionated organic-solvent soluble preparation from chloroplasts were used in a45Ca++-ligand blot assay known to detect high affinity Ca++-binding sites in proteins.

S. D. Zakharov; R. G. Ewy; R. A. Dilley



Complexation of bisphosphonates with ytterbium(III): application of phosphate and ATP detection assay based on Yb(3+)-pyrocatechol violet.  


The coordination chemistry of bisphosphonates with Yb(3+) was investigated to evaluate the potential of the UV-vis based detection method using the Yb(3+)-pyrocatechol complexation reaction as a sensor for bisphosphonates. The complexation chemistry of Yb(3+) with phosphate and ATP analogs was previously described (E. Gaidamauskas, K. Saejueng, A.A. Holder, S. Bharuah, B.A. Kashemirov, D.C. Crans, C.E. McKenna, J. Biol. Inorg. Chem. 13 (2008) 1291-1299), and we here studied the complexation chemistry of bisphosphonates in this system. The spectrophotometric assay yields direct evidence for formation of a 4:3 metal to ligand complex at neutral pH. Direct evidence for Yb(3+):methylenebis(phosphonate) complexes with 1:1 and 1:2 stoichiometry was also obtained by potentiometry at acidic and basic pH. Direct evidence for complex formation was obtained using (1)H NMR spectroscopy although the stoichiometry was not accessed at neutral pH. Our results suggest that the spectroscopic observation of the YbPV complex can be used to conveniently measure concentrations of bisphosphonates down to 2-3 microM. PMID:19850352

Gaidamauskas, Ernestas; Parker, Helen; Kashemirov, Boris A; Holder, Alvin A; Saejueng, Kanokkarn; McKenna, Charles E; Crans, Debbie C



Complexation of bisphosphonates with Ytterbium(III): Application of phosphate and ATP detection assay based on Yb3+-pyrocatechol violet  

PubMed Central

The coordination chemistry of bisphosphonates with Yb3+ was investigated to evaluate the potential of the UV-Vis based detection method using the Yb3+-pyrocatechol complexation reaction as a sensor for bisphosphonates. The complexation chemistry of Yb3+ with phosphate and ATP analogs was previously described (E. Gaidamauskas et al J. Biol. Inorg. Chem. 13 (2008) 1291-1299), and we here study the complexation chemistry of bisphosphonates in this system. The spectrophotometric assay yields direct evidence for formation of a 4:3 metal to ligand complex at neutral pH. Direct evidence for Yb3+ : methylenebis(phosphonate) complexes with 1:1 and 1:2 stoichiometry was also obtained by potentiometry at acidic and basic pH. Direct evidence for complex formation was obtained using 1H NMR spectroscopy although the stoichiometry was not accessed at neutral pH. Our results suggest that the spectroscopic observation of the YbPV complex can be used to conveniently measure concentrations of bisphosphonates down to 2-3 ?M .

Gaidamauskas, Ernestas; Parker, Helen; Kashemirov, Boris A.; Holder, Alvin A.; Saejueng, Kanokkarn; McKenna, Charles E.; Crans, Debbie C.



Definition of All Weather Landing System Requirements for Category III Operation in Corvair 880 n 112.  

National Technical Information Service (NTIS)

Studies were made to define an All-Weather Landing System (AWLS) to be used to establish requirements for operation in Category III weather minimums. Initially a limited (Category II) system will be installed with automatic VFR touchdown capability which ...



Assessment of severe malaria in a multicenter, phase III, RTS, S/AS01 malaria candidate vaccine trial: case definition, standardization of data collection and patient care  

PubMed Central

Background An effective malaria vaccine, deployed in conjunction with other malaria interventions, is likely to substantially reduce the malaria burden. Efficacy against severe malaria will be a key driver for decisions on implementation. An initial study of an RTS, S vaccine candidate showed promising efficacy against severe malaria in children in Mozambique. Further evidence of its protective efficacy will be gained in a pivotal, multi-centre, phase III study. This paper describes the case definitions of severe malaria used in this study and the programme for standardized assessment of severe malaria according to the case definition. Methods Case definitions of severe malaria were developed from a literature review and a consensus meeting of expert consultants and the RTS, S Clinical Trial Partnership Committee, in collaboration with the World Health Organization and the Malaria Clinical Trials Alliance. The same groups, with input from an Independent Data Monitoring Committee, developed and implemented a programme for standardized data collection. The case definitions developed reflect the typical presentations of severe malaria in African hospitals. Markers of disease severity were chosen on the basis of their association with poor outcome, occurrence in a significant proportion of cases and on an ability to standardize their measurement across research centres. For the primary case definition, one or more clinical and/or laboratory markers of disease severity have to be present, four major co-morbidities (pneumonia, meningitis, bacteraemia or gastroenteritis with severe dehydration) are excluded, and a Plasmodium falciparum parasite density threshold is introduced, in order to maximize the specificity of the case definition. Secondary case definitions allow inclusion of co-morbidities and/or allow for the presence of parasitaemia at any density. The programmatic implementation of standardized case assessment included a clinical algorithm for evaluating seriously sick children, improvements to care delivery and a robust training and evaluation programme for clinicians. Conclusions The case definition developed for the pivotal phase III RTS, S vaccine study is consistent with WHO recommendations, is locally applicable and appropriately balances sensitivity and specificity in the diagnosis of severe malaria. Processes set up to standardize severe malaria data collection will allow robust assessment of the efficacy of the RTS, S vaccine against severe malaria, strengthen local capacity and benefit patient care for subjects in the trial. Trial registration NCT00866619



The Impact of Extent and Location of Mediastinal Lymph Node Involvement on Survival in Stage III Non-Small Cell Lung Cancer Patients Treated With Definitive Radiotherapy  

SciTech Connect

Purpose: Several surgical series have identified subcarinal, contralateral, and multilevel nodal involvement as predictors of poor overall survival in patients with Stage III non-small-cell lung cancer (NSCLC) treated with definitive resection. This retrospective study evaluates the impact of extent and location of mediastinal lymph node (LN) involvement on survival in patients with Stage III NSCLC treated with definitive radiotherapy. Methods and Materials: We analyzed 106 consecutive patients with T1-4 N2-3 Stage III NSCLC treated with definitive radiotherapy at University of Pennsylvania between January 2003 and February 2009. For this analysis, mediastinal LN stations were divided into four mutually exclusive groups: supraclavicular, ipsilateral mediastinum, contralateral mediastinum, and subcarinal. Patients' conditions were then analyzed according to the extent of involvement and location of mediastinal LN stations. Results: The majority (88%) of patients received sequential or concurrent chemotherapy. The median follow-up time for survivors was 32.6 months. By multivariable Cox modeling, chemotherapy use (hazard ratio [HR]: 0.21 [95% confidence interval (CI): 0.07-0.63]) was associated with improved overall survival. Increasing primary tumor [18F]-fluoro-2-deoxy-glucose avidity (HR: 1.11 [CI: 1.06-1.19]), and subcarinal involvement (HR: 2.29 [CI: 1.11-4.73]) were significant negative predictors of overall survival. On univariate analysis, contralateral nodal involvement (HR: 0.70 [CI: 0.33-1.47]), supraclavicular nodal involvement (HR: 0.78 [CI: 0.38-1.67]), multilevel nodal involvement (HR: 0.97 [CI: 0.58-1.61]), and tumor size (HR: 1.04 [CI: 0.94-1.14]) did not predict for overall survival. Patients with subcarinal involvement also had lower rates of 2-year nodal control (51.2% vs. 74.9%, p = 0.047) and 2-year distant control (28.4% vs. 61.2%, p = 0.043). Conclusions: These data suggest that the factors that determine oncologic outcome in Stage III NSCLC patients treated with definitive radiotherapy are distinct from those observed in patients who undergo surgical resection. The ultimate efficacy of radiation in locally advanced NSCLC is dependent on the intrinsic biology of the tumor.

Fernandes, Annemarie T. [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA (United States); Mitra, Nandita [Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA (United States); Xanthopoulos, Eric [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA (United States); Evans, Tracey; Stevenson, James; Langer, Corey [Department of Medical Oncology, University of Pennsylvania, Philadelphia, PA (United States); Kucharczuk, John C. [Department of Thoracic Surgery, University of Pennsylvania, Philadelphia, PA (United States); Lin, Lilie; Rengan, Ramesh [Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA (United States)



Molecular Structure of ATP  

NSDL National Science Digital Library

In plant cells, ATP is produced in the cristae of mitochondria and chloroplasts. Christae are the multiply-folded inner membranes of a cell's mitochondrion, which are finger-like projections. The walls of the cristae are the site of the cell's energy production (it is where ATP is generated). Chloroplasts are made up of stacks of thylakoid disks that contain chlorophyll. Production of ATP molecules from sunlight takes place on thylakoid disks. The mechanism of ATP synthesis is the same in both mitochondria and chloroplasts. An important role of ATP as a plant molecule is to provide energy for biosynthesis. Interestingly enough, this chemical energy can also be converted into light energy in the reaction catalyzed by luciferase. Each molecule of ATP consumed in the reaction produces one photon of light.



Type III and type IV endoleak: toward a complete definition of blood flow in the sac after endoluminal AAA repair.  


In this document the authors continue to refine their seminal categorization of endoleak, a major complication of endovascular aneurysm repair. In addition to type I (related to the graft device itself) and type II (retrograde flow from collateral branches) endoleak, they propose two new categories: endoleak due to fabric tears, graft disconnection, or disintegration would be classified type III, and flow through the graft presumed to be associated with graft wall "porosity" would be categorized as type IV endoleak. PMID:9867318

White, G H; May, J; Waugh, R C; Chaufour, X; Yu, W



ATP signalling in epilepsy  

PubMed Central

This paper focuses on a role for ATP neurotransmission and gliotransmission in the pathophysiology of epileptic seizures. ATP along with gap junctions propagates the glial calcium wave, which is an extraneuronal signalling pathway in the central nervous system. Recently astrocyte intercellular calcium waves have been shown to underlie seizures, and conventional antiepileptic drugs have been shown to attenuate these calcium waves. Blocking ATP-mediated gliotransmission, therefore, represents a potential target for antiepileptic drugs. Furthermore, while knowledge of an antiepileptic role for adenosine is not new, a recent study showed that adenosine accumulates from the hydrolysis of accumulated ATP released by astrocytes and is believed to inhibit distant synapses by acting on adenosine receptors. Such a mechanism is consistent with a surround-inhibitory mechanism whose failure would predispose to seizures. Other potential roles for ATP signalling in the initiation and spread of epileptiform discharges may involve synaptic plasticity and coordination of synaptic networks. We conclude by making speculations about future developments.

Tolias, Christos M.; Burnstock, Geoffrey



Electron transfer precedes ATP hydrolysis during nitrogenase catalysis  

PubMed Central

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s?1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s?1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s?1, 25 °C), (iii) Phosphate release (kPi = 16 s?1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s?1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein.

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.



Hepatocellular ATP-binding cassette protein expression enhances ATP release and autocrine regulation of cell volume.  


In a model liver cell line, recovery from swelling is mediated by a sensitive autocrine pathway involving conductive release of ATP, P2 receptor stimulation, and opening of membrane Cl- channels (Wang, Y., Roman, R. M., Lidofsky, S. D., and Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release are not known. Based on evidence that certain ATP-binding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ABC proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins. In both cell types, Cl- current activation (ICl-swell) and volume recovery following swelling were dependent on conductive ATP efflux. The rate of volume recovery was approximately 6-fold faster in HTC-R cells compared with HTC cells. This effect is likely due to enhanced ABC protein-dependent ATP release since (i) ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 microM verapamil and 15 microM cyclosporin A); (ii) swelling-induced Cl- current density was similar in both cell types (approximately -50 pA/pF; not significant); and (iii) ATP conductance measured by whole-cell techniques was increased approximately 3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells exhibited enhanced survival during hypotonic stress. By modulating ATP release, hepatic ABC proteins may play a key role in the cellular pathways coupling changes in cell volume to ion permeability and secretion. PMID:9268333

Roman, R M; Wang, Y; Lidofsky, S D; Feranchak, A P; Lomri, N; Scharschmidt, B F; Fitz, J G



Adenosine and ATP Receptors  

Microsoft Academic Search

Adenosine and ATP, via P1 and P2 receptors respectively, can modulate pain transmission under physiological, inflammatory,\\u000a and neuropathic pain conditions. Such influences reflect peripheral and central actions and effects on neurons as well as\\u000a other cell types. In general, adenosine A1 receptors produce inhibitory effects on pain in a number of preclinical models\\u000a and are a focus of attention. In

J. Sawynok


How ATP inhibits the open K(ATP) channel.  


ATP-sensitive potassium (K(ATP)) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 microM. PMID:18591420

Craig, Tim J; Ashcroft, Frances M; Proks, Peter



Optogenetic control of ATP release  

NASA Astrophysics Data System (ADS)

Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.



ATP release via anion channels.  


ATP serves not only as an energy source for all cell types but as an 'extracellular messenger' for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg(2+) and/or H(+) salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP(4-) in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed. PMID:18404516

Sabirov, Ravshan Z; Okada, Yasunobu



Cervical anterior transpedicular screw fixation (ATPS)--Part II. Accuracy of manual insertion and pull-out strength of ATPS.  


Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3-T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of "critical" pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm(2) and a mean osseus screw purchase of 27.2 mm was 467.8 N. In comparison, POS of 22 VBS screws with a mean BMD of 0.533 g/cm(2) and a mean osseus screw purchase of 16.0 mm was 181.6 N. The difference in ultimate pull-out strength between the ATPS and VBS group was significant (p < 0.000001). Also, accuracy of ATPS placement in axial plane was shown to be significantly correlated with POS. In contrast, there was no correlation between screw-length, BMD, or level of insertion and the POS of ATPS or VBS. The study demonstrated that the use of ATPS might be a new technique worthy of further investigation. The use of ATPS shows the potential to increase construct rigidity in terms of screw-plate pull-out resistance. It might diminish construct failures during anterior-only reconstructions of the highly unstable decompressed cervical spine. PMID:18224357

Koller, Heiko; Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kässmann, H; Resch, Herbert; Hitzl, Wolfgang



Cervical anterior transpedicular screw fixation (ATPS)--Part II. Accuracy of manual insertion and pull-out strength of ATPS  

PubMed Central

Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3–T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of “critical” pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm2 and a mean osseus screw purchase of 27.2 mm was 467.8 N. In comparison, POS of 22 VBS screws with a mean BMD of 0.533 g/cm2 and a mean osseus screw purchase of 16.0 mm was 181.6 N. The difference in ultimate pull-out strength between the ATPS and VBS group was significant (p < 0.000001). Also, accuracy of ATPS placement in axial plane was shown to be significantly correlated with POS. In contrast, there was no correlation between screw-length, BMD, or level of insertion and the POS of ATPS or VBS. The study demonstrated that the use of ATPS might be a new technique worthy of further investigation. The use of ATPS shows the potential to increase construct rigidity in terms of screw-plate pull-out resistance. It might diminish construct failures during anterior-only reconstructions of the highly unstable decompressed cervical spine. Electronic supplementary material The online version of this article (doi:10.1007/s00586-007-0573-x) contains supplementary material, which is available to authorized users.

Acosta, Frank; Tauber, Mark; Fox, Michael; Martin, Hudelmaier; Forstner, Rosmarie; Augat, Peter; Penzkofer, Rainer; Pirich, Christian; Kassmann, H.; Resch, Herbert; Hitzl, Wolfgang



ATP-dependent Chromatin Remodelling  

Microsoft Academic Search

Alterations of chromatin structure play an important role in gene regulation. One way of doing so involves ATP-dependent chromatin\\u000a remodelling enzymes that act as molecular machines coupling ATP-hydrolysis to structural changes of the nucleosome. Several\\u000a recent studies shed important insights into the mechanism of these factors and indicate that they couple DNA translocation\\u000a within the nucleosome to DNA loop propagation

Parul Choudhary; Patrick Varga-Weisz


Implications of Recent Clinical Trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines  

Microsoft Academic Search

The Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program issued an evidence-based set of guidelines on cholesterol management in 2001. Since the publication of ATP III, 5 major clinical trials of statin therapy with clinical end points have been published. These trials addressed issues that were not examined in previous clinical trials of cholesterol-lowering therapy. The

Scott M. Grundy; James I. Cleeman; C. Noel Bairey Merz; H. Bryan Brewer Jr; Luther T. Clark; Donald B. Hunninghake; Richard C. Pasternak; Sidney C. Smith Jr; Neil J. Stone



Bringing Definitions into High Definition  

ERIC Educational Resources Information Center

Why do definitions play such a central role in mathematics? It may seem obvious that precision about the terms one uses is necessary in order to use those terms reasonably (while reasoning). Definitions are chosen so as to be definite about the terms one uses, but also to make both the statement of, and the reasoning to justify, theorems as…

Mason, John



ATP and ADP Actin States  

PubMed Central

This minireview is dedicated to the memory of Henryk Eisenberg and honors his major contributions to many areas of biophysics and to the analysis of macromolecular states and interactions in particular. This work reviews the ATP and ADP states of a ubiquitous protein, actins, and considers the present evidence for and against unique, nucleotide-dependent conformations of this protein. The effects of ATP and ADP on specific structural elements of actins, its loops and clefts, as revealed by mutational, crosslinking, spectroscopic, and EPR methods are discussed. It is concluded that the existing evidence points to dynamic equilibria of these structural elements among various conformational states in both ATP- and ADP-actins, with the nucleotides impacting the equilibria distributions.

Kudryashov, Dmitri S.; Reisler, Emil



[Dysperception of body image and dysmorphophobias in mental anorexia. Apropos of 115 cases involving both sexes. III. Physiopathogenic deductions and introduction of a novel definition of the disease].  


Ignoring of emaciation (IE), fear of any weight recovery (Dalpha), and dismorphophobias (DPP) represent the central problem of AN, with different incidence. Fundamental need of being lean expresses, at lesss in girls, distress of personality insufficiently prepared to autonomous adult life, with its responsabilities. Obesity-DPP may correspond to projection upon the body of the obsessing conviction of being inferior, with regard to social and publicitary patterns, and get an active play in starting and management of weight loss. So AN is either an attempt to accomodate this critic situation, trying to incarnate actual female archetype, either, in the more severe cases, a renouncing with an obstinate physical and psychological recession to the state of a protected child. It seems to correspond to an attempt of negation of morbid character of this situation, so that it may be perpetuated and so that feeling of culpability can be decreased in front of familial recrimination. Constancy of these symptoms, and their relation with deep meaning of this illness, justify their introduction into a new definition of AN, diagnosed by association of at less 2 out of 3 major criterious (loss of weight superior to 10% premorbid weight, feed restrictions and Dalpha) and one out of 2 minor criterions (amenorrhea and IE). PMID:727618

Buvat, J; Buvat-Herbaut, M



Oral bioavailability of ATP after prolonged administration.  


Purinergic receptors are important for the regulation of inflammation, muscle contraction, neurotransmission and nociception. Extracellular ATP and its metabolites are the main ligands for these receptors. Occasional reports on beneficial results of ATP administration in human and animal studies have suggested the bioavailability of oral ATP supplements. We investigated whether prolonged daily intake of oral ATP is indeed bioavailable. Thirty-two healthy subjects were randomised to receive 0, 250, 1250 or 5000 mg ATP per d for 28 d by means of enteric-coated pellets. In addition, on days 0 and 28, all thirty-two subjects received 5000 mg ATP to determine whether prolonged administration would induce adaptations in the bioavailability of ATP. ATP supplementation for 4 weeks did not lead to changes in blood or plasma ATP concentrations. Of all ATP metabolites, only plasma uric acid levels increased significantly after the administration of 5000 mg of ATP. Prolonged administration of ATP was safe as evidenced from liver and kidney parameters. We conclude that oral administration of ATP only resulted in increased uric acid concentrations. On the basis of these findings, we seriously question the claimed efficacy of oral ATP at dosages even lower than that used in the present study. PMID:21129239

Coolen, Erik J C M; Arts, Ilja C W; Bekers, Otto; Vervaet, Chris; Bast, Aalt; Dagnelie, Pieter C



Definitely Life but not Definitively  

NASA Astrophysics Data System (ADS)

Although there have been attempts at a definition of life from many disciplines, none is accepted by all as definitive. Some people believe that it is impossible to define ‘life’ adequately at the moment. We agree with this point of view on linguistic grounds, examining the different types of definition, the contexts in which they are used and their relative usefulness as aids to arriving at a scientific definition of life. We look at some of the more recent definitions and analyse them in the light of our criteria for a good definition. We argue that since there are so many linguistic and philosophical difficulties with such a definition of life, what is needed is a series of working descriptions, which are suited to the audience and context in which they are used and useful for the intended purpose. We provide some ideas and examples of the forms these may take.

Oliver, Joan D.; Perry, Randall S.



'Domino' systems biology and the 'A' of ATP.  


We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP. PMID:23031542

Verma, Malkhey; Zakhartsev, Maksim; Reuss, Matthias; Westerhoff, Hans V



Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation  

PubMed Central

Transient accumulation of extracellular ATP reflects both release of ATP from intracellular stores and altered rates of ATP metabolism by ecto-enzymes. Ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) and ecto-nucleotide pyrophosphatases (eNPPs) degrade ATP, while ecto-nucleotide diphosphokinases (eNDPKs) synthesize ATP from ambient ADP. Although the methylene ATP analogs ??-meATP and ??-meATP are widely used as metabolically stable tools for the analysis of purinergic signaling, their specific effects on eNTPDase, eNPP, and eNDPK activities have not been defined. This study compared the actions of these analogs on extracellular ATP metabolism by human 1321N1 astrocytes, rat PC12 pheochomocytoma cells, and rat C6 glioma cells. Both analogs significantly reduced clearance of extracellular ATP by 1321N1 cells that express both eNTPDases and eNPPs, as well as by C6 cells that exclusively express eNPPs. In contrast, both analogs were much less efficacious in inhibiting ATP clearance by PC12 cells that predominantly express eNTPDases. ??-meATP, but not ??-meATP, was effectively hydrolyzed by the 1321N1 and C6 cells; PC12 cells did not significantly degrade this analog. ??-meATP, but not ??-meATP, acted as a substrate for purified yeast NDPK to generate ATP via trans-phosphorylation of ADP. ??-meATP also acted as substrate for the eNDPK activities expressed by 1321N1, PC12, and C6 cells and thereby induced extracellular ATP accumulation in the presence of ambient or exogenously added ADP. These results indicate that methylene ATP analogs exert complex and cell-specific effects on extracellular ATP metabolism that can significantly modify interpretation of studies that use these reagents as probes of purinergic signal transduction in intact tissues.

Joseph, Sheldon M; Pifer, Matthew A; Przybylski, Ronald J; Dubyak, George R



20 CFR 631.87 - Definitions.  

Code of Federal Regulations, 2012 CFR

...EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROGRAMS UNDER TITLE III OF THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.87 Definitions. As used in this subpart, the term unit of general local...



20 CFR 631.87 - Definitions.  

Code of Federal Regulations, 2010 CFR

...EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROGRAMS UNDER TITLE III OF THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.87 Definitions. As used in this subpart, the term unit of general local...



20 CFR 631.87 - Definitions.  

Code of Federal Regulations, 2011 CFR

...EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROGRAMS UNDER TITLE III OF THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.87 Definitions. As used in this subpart, the term unit of general local...



77 FR 39626 - Further Definition of “Swap Dealer,” “Security-Based Swap Dealer,” “Major Swap Participant...  

Federal Register 2010, 2011, 2012, 2013 the third column, correct paragraph (hhh)(6)(iii)(B)(2) to read as follows: Sec. 1.3 Definitions. * * * * * (hhh) * * * (6) * * * (iii) * * * (B...the amount calculated under paragraph (hhh)(6)(iii)(B)(1) of this...



Circadian regulation of ATP release in astrocytes  

PubMed Central

Circadian clocks sustain daily oscillations in gene expression, physiology and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP3 signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP3 signaling to express daily rhythms in ATP release.

Marpegan, Luciano; Swanstrom, Adrienne E.; Chung, Kevin; Simon, Tatiana; Haydon, Philip G.; Khan, Sanjoy K.; Liu, Andrew C.; Herzog, Erik D.; Beaule, Christian



Extracellular ATP in Plants. Visualization, Localization, and Analysis of Physiological Significance in Growth and Signaling1[W  

PubMed Central

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (??meATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.

Kim, Sung-Yong; Sivaguru, Mayandi; Stacey, Gary



25 CFR 291.2 - Definitions  

Code of Federal Regulations, 2011 CFR

...2011-04-01 false Definitions 291.2 Section 291.2 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR ECONOMIC ENTERPRISES CLASS III GAMING PROCEDURES § 291.2 Definitions (a) All terms have the same meaning as set...



ATP metabolism in skeletal muscle arterioles  

PubMed Central

Abstract The purpose of this study was to investigate the metabolism of Adenosine triphosphate (ATP) in skeletal muscle resistance arterioles and to determine whether this metabolism is altered during the rapid growth phase of the rat. We attempted to quantify ATP metabolism in gastrocnemius first?order arterioles from 8?, 10?, and 12?week?old rats. We measured ATP metabolism using an ATPase/GTPase assay with whole vessel segments as well as using a real?time adenosine biosensor following electric field stimulation. Our first method of measuring ATP metabolism allowed us to measure the amount of free phosphate produced with ATP as a substrate. When ecto?nucleotidase activity was inhibited by ARL67156, pyridoxal phosphate?6?azophenly?2?, 4??disulfonic acid (PPADS), or suramin prior to adding ATP, we found that the rate of phosphate production was significantly reduced by 27%, 21%, and 22%, respectively (P < 0.05). Our second method of measuring ATP metabolism allowed us to measure the amount of adenosine produced following electric field stimulation of the arteriole with and without nucleotidase inhibitors. Surprisingly, we found that adenosine overflow was not attenuated by nucleotidase inhibitors. We concluded that ecto?phosphodieterase/phyrophophatase (E?NPP), ecto?diadenosine polyphosphatase (ApnA), NTPDase1 and 2, and E5NT may be present on the gastrocnemius 1A arteriole and do play a role in ATP metabolism. Between the ages of 8 weeks and 12 weeks, however, overall ATP metabolism may not change.

Stone, Audrey J.; Evanson, Kirk W.; Kluess, Heidi A.



Preservation of ATP in Hypersaline Environments  

PubMed Central

High concentrations of particulate ATP were found in the anoxic brines of the Orca Basin and East Flower Garden, Gulf of Mexico. Other measurements indicative of growth and respiration suggested that the microbial community in the brines was inactive, but somehow the ATP associated with the cells persisted. Conceivably, when cells growing just above the interface sank into the brine, the increased osmotic stress could elicit an osmoregulatory response resulting in increased ATP. It was also possible that hydrolytic enzymes were inactivated, resulting in the preservation of ATP. Experiments in which a culture of marine bacteria was suspended in menstrua of different salinities comparable to those found across the Orca Basin interface revealed that as salinity increased, ATP increased three- to sixfold. Within 24 h the ATP fell to its initial level and remained at that concentration for 3 days, at which time the experiment was terminated. In contrast, the control suspensions, at a salinity of 28% (grams per liter) had 1/10th of the initial ATP concentration when the experiment was ended. Cells were also exposed to killing UV irradiation, enabling us to demonstrate with absolute certainty that cellular ATP could be preserved. At the end of the experiment, the viable component of the population was reduced by orders of magnitude by UV irradiation, but the ATP levels of the cells suspended in brine did not decrease. In certain environments it appears that the conventional analytical tools of the microbial ecologist must be interpreted with caution.

Tuovila, Bruce J.; Dobbs, Fred C.; LaRock, Paul A.; Siegel, B. Z.



Catalytic strategy used by the myosin motor to hydrolyze ATP.  


Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose ?-phosphate is not in the previously reported HP?O4 (2-) state, but in the H2P?O4 (-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the ?-phosphate of ATP in a dissociated metaphosphate (P?O3 (-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes. PMID:25006262

Kiani, Farooq Ahmad; Fischer, Stefan



Detection of in vitro kinase generated protein phosphorylation sites using gamma[18O4]-ATP and mass spectrometry.  


A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [gamma((18)O4)-ATP], has been developed. The ability to use gamma((18)O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of gamma((18)O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and gamma((18)O4)-ATP. Standard peptides phosphorylated using gamma((18)O4)-ATP via in vitro kinase reactions showed no exchange loss of (18)O with (16)O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins. PMID:17877366

Zhou, Ming; Meng, Zhaojing; Jobson, Andrew G; Pommier, Yves; Veenstra, Timothy D



Enzymatic Treatment to Eliminate the Extracellular ATP for Improving the Detectability of Bacterial Intracellular ATP  

Microsoft Academic Search

A novel and effective treatment of biological samples with a combination of adenosine phosphate deaminase and apyrase was developed for reducing extracellular ATP, which has been a major problem encountered in improving the sensitivity of assays for intracellular ATP by the firefly luciferin–luciferase (L-L) method. Under the enzymatic reaction conditions, ATP and the related adenosine derivatives were converted to IMP,

Tatsuya Sakakibara; Seiji Murakami; Noriaki Hattori; Moto-o Nakajima; Kazuhiro Imai



Sequential (gemcitabine/vinorelbine) and concurrent (gemcitabine) radiochemotherapy with FDG-PET-based target volume definition in locally advanced non-small cell lung cancer: first results of a phase I/II study  

PubMed Central

Background The aim of the study was to determine the maximal tolerated dose (MTD) of gemcitabine every two weeks concurrent to radiotherapy, administered during an aggressive program of sequential and simultaneous radiochemotherapy for locally advanced, unresectable non-small cell lung cancer (NSCLC) and to evaluate the efficacy of this regime in a phase II study. Methods 33 patients with histologically confirmed NSCLC were enrolled in a combined radiochemotherapy protocol. 29 patients were assessable for evaluation of toxicity and tumor response. Treatment included two cycles of induction chemotherapy with gemcitabine (1200 mg/m2) and vinorelbine (30 mg/m2) at day 1, 8 and 22, 29 followed by concurrent radiotherapy (2.0 Gy/d; total dose 66.0 Gy) and chemotherapy with gemcitabine every two weeks at day 43, 57 and 71. Radiotherapy planning included [18F] fluorodeoxyglucose positron emission tomography (FDG PET) based target volume definition. 10 patients were included in the phase I study with an initial gemcitabine dose of 300 mg/m2. The dose of gemcitabine was increased in steps of 100 mg/m2 until the MTD was realized. Results MTD was defined for the patient group receiving gemcitabine 500 mg/m2 due to grade 2 (next to grade 3) esophagitis in all patients resulting in a mean body weight loss of 5 kg (SD = 1.4 kg), representing 8% of the initial weight. These patients showed persisting dysphagia 3 to 4 weeks after completing radiotherapy. In accordance with expected complications as esophagitis, dysphagia and odynophagia, we defined the MTD at this dose level, although no dose limiting toxicity (DLT) grade 3 was reached. In the phase I/II median follow-up was 15.7 months (4.1 to 42.6 months). The overall response rate after completion of therapy was 64%. The median overall survival was 19.9 (95% CI: [10.1; 29.7]) months for all eligible patients. The median disease-free survival for all patients was 8.7 (95% CI: [2.7; 14.6]) months. Conclusion After induction chemotherapy, the maximum tolerated dose and frequency of gemcitabine was defined at 500 mg/m2 every two weeks in three cycles during a maximum of 7 weeks of thoracic radiotherapy for the phase II study. This regimen represents an effective and tolerable therapy in the treatment of NSCLC.

Gagel, Bernd; Piroth, Marc; Pinkawa, Michael; Reinartz, Patrick; Krohn, Thomas; Kaiser, Hans J; Stanzel, Sven; Breuer, Christian; Asadpour, Branka; Schmachtenberg, Axel; Eble, Michael J




NSDL National Science Digital Library

Plasma membrane calcium-transporting ATPase 4 (ATP2B4), also known as A0114, is a multi-pass membrane protein, which catalyzes the hydrolysis of adenosine triphosphate (ATP) coupled with the transport of calcium out of the cell.



Chemotherapy induces ATP release from tumor cells.  


Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies. PMID:19855167

Martins, Isabelle; Tesniere, Antoine; Kepp, Oliver; Michaud, Mickael; Schlemmer, Frederic; Senovilla, Laura; Séror, Claire; Métivier, Didier; Perfettini, Jean-Luc; Zitvogel, Laurence; Kroemer, Guido



Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia.  


Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes. PMID:22133679

Kunugi, Satohiko; Iwabuchi, Sadahiro; Matsuyama, Daisuke; Okajima, Takaharu; Kawahara, Koichi



Expression of the non-gastric H+/K+ ATPase ATP12A in normal and pathological human prostate tissue.  


Altered cellular proton handling and cell volume regulation are hallmarks of tumorigenesis. To investigate a possible involvement of the non-gastric H(+)/K(+) ATPase ATP12A (ATP1AL1) in prostate cancer, we performed immunohistochemistry in formalin-fixed, paraffin-embedded histological sections from benign and malignant human prostate lesions. Normal prostate tissue displayed a membrane-bound ATP12A staining with focal accumulated pattern, whereas in the benign prostate hyperplasia (BPH) and cancerous prostate tissue (tumor grade I-III) the protein appears to be displaced in the luminal cells of the glandular epithelium. Hence, the expression pattern of ATP12A is markedly altered in BPH and prostate cancer. To test for altered gene expression of ATP12A we performed quantitative reverse transcriptase PCR (QRT-PCR) in normal (tumor-free) prostate tissue, BPH and tumor stages I-III using a prostate cancer cDNA array. However, no significantly different expression levels could be detected in the various disease states compared to normal tissue, which contrasts the findings from immunohistochemistry and points to the possibility of altered post-translational processing and/or sorting of the protein. We further show that ATP12A mRNA is expressed at different levels in PC-3 and LNCaP prostate cancer cells, with a significant ~26-fold higher expression in the latter cell type. Protein expression in these tumor cell lines was verified by Western blot. PMID:22179016

Streif, Doris; Iglseder, Eva; Hauser-Kronberger, Cornelia; Fink, Klaus G; Jakab, Martin; Ritter, Markus



The Rotary Mechanism of the ATP Synthase  

PubMed Central

The FOF1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous FO sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium.

Nakamoto, Robert K.; Scanlon, Joanne A. Baylis; Al-Shawi, Marwan K.



The Role of ATP in Sleep Regulation  

PubMed Central

One of the functions of sleep is to maintain energy balance in the brain. There are a variety of hypotheses related to how metabolic pathways interact with sleep/wake regulation. A major finding that demonstrates an interaction between sleep and metabolic homeostasis is the involvement of adenosine in sleep homeostasis. An accumulation of adenosine is supplied from ATP, which can act as an energy currency in the cell. Extracellularly, ATP can act as an activity-dependent signaling molecule, especially in regard to communication between neurons and glia, including astrocytes. Furthermore, the intracellular AMP/ATP ratio controls the activity of AMP-activated protein kinase, which is a potent energy regulator and is recently reported to play a role in the regulation of sleep homeostasis. Brain ATP may support multiple functions in the regulation of the sleep/wake cycle and sleep homeostasis.

Chikahisa, Sachiko; Sei, Hiroyoshi



Target-induced structure switching of hairpin aptamers for label-free and sensitive fluorescent detection of ATP via exonuclease-catalyzed target recycling amplification.  


In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a "mix-and-detect" fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules. PMID:23974161

Xu, Yunying; Xu, Jin; Xiang, Yun; Yuan, Ruo; Chai, Yaqin



Mechanisms for ATP-dependent chromatin remodelling  

Microsoft Academic Search

During the past year, major advances have been made towards understanding the function of ATP-dependent chromatin-remodelling activities both in vitro and in vivo. These suggest that ATP-dependent chromatin-remodelling activities are capable of both altering the structure of individual nucleosomes and acting in concert with other forms of chromatin-modifying enzymes, to regulate the formation and decondensation of chromatin fibres.

Andrew Flaus; Tom Owen-Hughes



Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells.  


ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target. PMID:24973521

Qian, Yanrong; Wang, Xuan; Liu, Yi; Li, Yunsheng; Colvin, Robert A; Tong, Lingying; Wu, Shiyong; Chen, Xiaozhuo



The nodulin vfENOD18 is an ATP-binding protein in infected cells of Vicia faba L. nodules.  


Recently we described the novel nodulin gene VfENOD18, whose corresponding transcripts were restricted to the nitrogen-fixing zone III of broad bean root nodules. To characterize VfENOD18 on the protein level, polyclonal antibodies were generated using the purified recombinant VfENOD18 protein produced in Escherichia coli by employing the pMAL-c expression system. These antibodies recognized immunoreactive proteins isolated from indeterminate nodules of different leguminous plants, but also from non-symbiotic tissues of Glycine max and from tissues of Arabidopsis thaliana and Zea mays. Using immunogold labelling the nodulin VfENOD18 was localized to the cytoplasm of infected cells in the nitrogen-fixing zone of broad bean nodules. Due to the homology of the VfENOD18 sequence to that of the ATP-binding protein MJ0577 from the hyperthermophile Methanococcus jannaschii the recombinant VfENOD18 protein was tested for ATP-binding. Using the biotin photoaffinity ATP analogue 8N3ATP[gamma]biotin it could be demonstrated that VfENOD18 is an ATP-binding protein. PCR experiments revealed that the amino acid sequences of the putative C-terminal ATP-binding sites of the VfENOD 18 homologues from Lens culinaris, Vicia hirsuta, Vicia sativa and Vicia villosa were conserved. We propose that VfENOD18 is a member of a novel family of ATP-binding proteins in plants. PMID:11785936

Becker, J D; Moreira, L M; Kapp, D; Frosch, S C; Pühler, A; Perlic, A M



Oxidant-mediated apoptosis in proximal tubular epithelial cells following ATP depletion and recovery.  


Oxidant-mediated apoptosis has been implicated in renal injury due to ischemia reperfusion (IR); however, the apoptotic signaling pathways following IR have been incompletely defined. The purpose of this study was to examine the role of oxidants on cell death in a model of in vitro simulated IR injury in renal proximal tubular epithelial cells by analyzing the effects of a cell-permeable superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTmPyP). Renal proximal tubular epithelial cells were ATP depleted for 2, 4, or 6 h, followed by 2 h of recovery. We found that MnTmPyP was effective in attenuating cytotoxicity (P<0.001) and decreasing steady-state oxidant levels (P<0.001) and apoptotic cell death (P<0.001) following ATP depletion-recovery. MnTmPyP treatment prevented the early cytosolic release of cytochrome c and increased Bcl-2 protein levels following short durations of ATP depletion-recovery. After longer periods of ATP depletion-recovery, we observed a significant increase in TNF-alpha protein levels (P<0.001) and caspase-8 activation (P<0.001), both of which were decreased (P<0.001) by treatment with MnTmPyP. Our results suggest that oxidant mediated apoptosis via the mitochondrial pathway during the early phase of ATP depletion and by activation of the receptor-mediated apoptotic pathway following longer durations of injury. PMID:17997382

Maenpaa, Cheryl J; Shames, Brian D; Van Why, Scott K; Johnson, Christopher P; Nilakantan, Vani



ATP Synthase Is Necessary for Microcin H47 Antibiotic Action  

PubMed Central

Microcin H47 is a gene-encoded peptide antibiotic produced by a natural Escherichia coli strain isolated in Uruguay. In order to identify cellular components necessary for its antibiotic action, microcin H47-resistant mutants isolated in this work, as well as previously described mutants affected in membrane proteins, were analyzed. These studies indicated that (i) receptor outer membrane proteins for ferric-catechol siderophores would be involved in microcin-specific binding to the cell surface, (ii) the TonB pathway is needed for microcin H47 uptake, and (iii) the presence of the ATP synthase complex is necessary for microcin action. The possibility that this last structure contains the antibiotic target is discussed.

Trujillo, Monica; Rodriguez, Eliana; Lavina, Magela



Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion.  


ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanisms remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on leg muscle interstitial ATP and norepinephrine (NE) concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and NE concentrations were measured during 1) femoral arterial ATP infusion (0.42 +/- 0.04 and 2.26 +/- 0.52 micromol/min; mean +/- SE) and 2) one-leg knee-extensor exercise (18 +/- 0 and 37 +/- 2 W) in 10 healthy men. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from approximately 0.3 l/min at baseline to 4.2 +/- 0.3 and 4.6 +/- 0.5 l/min, respectively, whereas it was reduced or unchanged in the control leg. During arterial ATP infusion, muscle interstitial ATP, ADP, AMP, and adenosine concentrations remained unchanged in both legs, but muscle interstitial NE increased from approximately 5.9 nmol/l at baseline to 8.3 +/- 1.2 and 8.7 +/- 0.7 nmol/l in the experimental and control leg, respectively (P < 0.05), in parallel to a reduction in arterial pressure (P < 0.05). During exercise, however, interstitial ATP, ADP, AMP, and adenosine concentrations increased in the contracting muscle (P < 0.05), but not in inactive muscle, whereas interstitial NE concentrations increased similarly in both active and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial purinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its local concentration. PMID:19797688

Mortensen, Stefan P; González-Alonso, José; Nielsen, Jens-Jung; Saltin, Bengt; Hellsten, Ylva



Ribonuclease III.  

National Technical Information Service (NTIS)

RNase III of Escherichia coli is the endonuclease responsible for the first steps in the post-transcriptional processing of E. coli ribosomal RNA. The purification and structure of RNase III are discussed. The action of RNase III in cleaving double-strand...

J. J. Dunn



ATP Sulfurylase from Higher Plants 1  

PubMed Central

ATP sulfurylase was purified extensively from green cabbage (Brassica capitata L.) leaf. The enzyme appears to be an asymmetric dimer composed of 57,000 dalton subunits. Initial velocity and product inhibition studies of the forward and reverse reactions point to an obligately ordered kinetic mechanism with MgATP adding before MoO42? (or SO42?). and MgPPi leaving before AMP + MoO42? (or adenosine-5?-phosphosulfate [APS]). The addition of excess purified fungal APS kinase to assay mixtures increased the rate of 35SO42? incorporation and MgPPi formation and extended the linearity of the forward reaction. This effect can be ascribed to the continual removal of APS, a potent product inhibitor of ATP sulfurylase. The specific activities of the enzyme in the APS synthesis, molybdolysis, MgATP synthesis, and sulfate-dependent [32P]-MgPPi-MgATP exchange assays were 3.3, 38, 38, and 4.3 micromole product formed per minute per milligram protein, respectively.

Osslund, Timothy; Chandler, Carol; Segel, Irwin H.



Inhibition of ATP Synthase by Chlorinated Adenosine Analogue  

PubMed Central

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing ? and ? subunits. Crystal structures of both ? and ? subunits that bind to the substrate, ADP, are known in tight binding (?dp?dp) and loose binding (?tp?tp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the ?tp?tp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (?dp?dp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 ?. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha



Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor-independent mechanisms  

PubMed Central

Periodate-oxidized ATP (oATP), which covalently modifies nucleotide-binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X7 nucleotide receptor (P2X7R) is irreversibly antagonized by oATP, it is unclear whether anti-inflammatory actions of oATP are predominantly mediated via its actions on P2X7R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X7R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes. oATP decreased by 40–70% the secretion of interleukin (IL)-8 stimulated by tumor necrosis factor-? (TNF-?) in all three cell types, by IL-1? in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF-?-stimulated IL-8 secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant P2X7R. oATP also attenuated cytokine-stimulated expression of nuclear factor-?B-luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of I?B. oATP had no effect on uridine triphosphate-induced activation of native P2Y2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y1 receptors. However, inhibition of P2Y1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF-?-stimulated IL-8 secretion. Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto-ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP. In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.

Beigi, Reza D; Kertesy, Sylvia B; Aquilina, Gretchen; Dubyak, George R



Sulfide-based ATP production in Urechis unicinctus  

NASA Astrophysics Data System (ADS)

We measured sulfide-based ATP production by isolated mitochondria from four tissues of Urechis unicinctus and the effects of inhibitors of respiratory complexes on ATP production were evaluated. The results show that these mitochondria could oxidize sulfide to produce ATP. The yield of sulfide-stimulated ATP varied from 5 nmol ATP/min/mg to 90 nmol ATP/min/mg according to the sulfide concentration and the source of the mitochondria. The maximum ATP synthesis occurred in hindgut mitochondria using 5 ?mol/L sulfide as a substrate. The effects of inhibitors (Rotenone, Antimycin A, Cyanide, and Salicylhydroxamic acid) on mitochondrial ATP production varied with the source of the mitochondria. Our results indicate that sulfide-based ATP production and the associated electron transport pathway are tissue-specific in U. unicinctus.

Ma, Zhuojun; Bao, Zhenmin; Wang, Sifeng; Zhang, Zhifeng



Metabolic differences in hippocampal 'Rett' neurons revealed by ATP imaging.  


Understanding metabolic control of neuronal function requires detailed knowledge of ATP handling in living neurons. We imaged ATP in organotypic hippocampal slices using genetically encoded sensor Ateam 1.03 modified to selectively transduce neurons in the tissue. ATP imaging indicated distinct differences in ATP production and consumption in dentate gyrus and cornu ammonis (CA) areas. Removal of extracellular Mg(2+) from the bath evoked epileptiform-like activity that was accompanied by ATP decline from 2-3 to 1-2mM. The slices fully recovered from treatment and showed persistent spontaneous activity. Neuronal discharges were followed by transient ATP changes and periodic activation of ATP-sensitive K(+) (K-ATP) channels. The biggest ATP decreases during epileptiform-like episodes of activity were observed in CA1 and CA3 neurons. Examination of neurons from the Rett model mice MeCP2(-/y) showed that seizure-like activity had earlier onset and subsequent spontaneous activity demonstrated more frequent discharges. Hippocampal MeCP2(-/y) neurons had higher resting ATP levels and showed bigger ATP decreases during epileptiform-like activity. More intense ATP turnover in MeCP2(-/y) neurons may result from necessity to maintain hippocampal function in Rett syndrome. Elevated ATP may make, in turn, Rett hippocampus more prone to epilepsy due to inadequate activity of K-ATP channels. PMID:24394521

Toloe, J; Mollajew, R; Kügler, S; Mironov, S L



ATP formation from adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analog.  


The purity of several preparations of adenyl-5'-yl imidodiphosphate (AMP-PNP) was analyzed using thin layer chromatography and the luciferin-luciferase assay. Three contaminants were identified: adenyl-5'-yl phosphoramidate, phosphorylated AMP-PNP, and ATP. The level of ATP contamination ranged from 0.02% to 0.3% in commercially obtained AMP-PNP preparations, and rose to 10% following incubation of AMP-PNP at 37 degrees C for 3 weeks in aqueous solution. The chemistry of the phosphoramidate bond is reviewed briefly, and evidence for a simple mechanism for the spontaneous formation of ATP from AMP-PNP is presented. PMID:7430085

Penningroth, S M; Olehnik, K; Cheung, A



Formal Definitions in Mathematics  

ERIC Educational Resources Information Center

The definition is an important language form in the register of mathematics. Students need to understand the structure of a definition so that they can make sense of the definitions they encounter and so that they can construct their own definitions as part of organising their thoughts about the concepts they have explored. This article suggests…

Shield, Mal



A reusable prepositioned ATP reaction chamber  

NASA Technical Reports Server (NTRS)

Luminescence biometer detects presence of life by means of light-emitting chemical reaction of luciferin and luciferase with adenosine triphosphate (ATP) that occurs in all living cells. Amount of light in reaction chamber is measured to determine presence and extent of life.

Hoffman, D. G.




NSDL National Science Digital Library

ATPase, sodium/potassium transporting, alpha 3 polypeptide (ATP1A3, also known as A0342) is a subunit of a cell membrane enzyme expressed in the brain that is a member of the family of cation transport ATPases (P-type).



Torque generation mechanism of ATP synthase  

NASA Astrophysics Data System (ADS)

ATP synthase is a rotary motor that produces adenosine triphosphate (ATP), the chemical currency of life. Our proposed electric field driven torque (EFT) model of FoF1-ATP synthase describes how torque, which scales with the number of c-ring proton binding sites, is generated by the proton motive force (pmf) across the mitochondrial inner membrane. When Fo is coupled to F1, the model predicts a critical pmf to drive ATP production. In order to fully understand how the electric field resulting from the pmf drives the c-ring to rotate, it is important to examine the charge distributions in the protonated c-ring and a-subunit containing the proton channels. Our calculations use a self-consistent field approach based on a refinement of reported structural data. The results reveal changes in pKa for key residues on the a-subunit and c-ring, as well as titration curves and protonation state energy diagrams. Health implications will be briefly discussed.

Miller, John; Maric, Sladjana; Scoppa, M.; Cheung, M.



Electric Field Driven Torque in ATP Synthase  

PubMed Central

FO-ATP synthase (FO) is a rotary motor that converts potential energy from ions, usually protons, moving from high- to low-potential sides of a membrane into torque and rotary motion. Here we propose a mechanism whereby electric fields emanating from the proton entry and exit channels act on asymmetric charge distributions in the c-ring, due to protonated and deprotonated sites, and drive it to rotate. The model predicts a scaling between time-averaged torque and proton motive force, which can be hindered by mutations that adversely affect the channels. The torque created by the c-ring of FO drives the ?-subunit to rotate within the ATP-producing complex (F1) overcoming, with the aid of thermal fluctuations, an opposing torque that rises and falls with angular position. Using the analogy with thermal Brownian motion of a particle in a tilted washboard potential, we compute ATP production rates vs. proton motive force. The latter shows a minimum, needed to drive ATP production, which scales inversely with the number of proton binding sites on the c-ring.

Miller, John H.; Rajapakshe, Kimal I.; Infante, Hans L.; Claycomb, James R.



Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.  


An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity. PMID:24872080

Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte




SciTech Connect

The direct detection of weakly interacting massive particles by a terrestrial device is widely recognised as a definitive proof of the cold dark matter hypothesis and a robust test of physics beyond the Standard Model. ZEPLIN-III is one of the latest generation of instruments specifically designed for this objective. This instrument has developed the two-phase liquid-gas xenon technology, and features high-field extraction, open plan geometry and low background components. Here we present the status of the project as of February 2008.

St Murphy, A. J. [School of Physics and Astronomy, University of Edinburgh, Edinburgh, EH9 3JZ (United Kingdom)



Findings from Advanced Technology Program's (ATP's) Survey of Joint Ventures.  

National Technical Information Service (NTIS)

The Advanced Technology Program (ATP) conducted a survey of all joint ventures that received an ATP award between 1991 and 2001. The survey was conducted to understand the motivations and impacts of joint venture collaborations. The findings show that the...

A. Wang J. O'Brien K. McTigue S. Shipp



Enhanced Anticancer Efficacy by ATP-Mediated Liposomal Drug Delivery.  


A liposome-based co-delivery system composed of a fusogenic liposome encapsulating ATP-responsive elements with chemotherapeutics and a liposome containing ATP was developed for ATP-mediated drug release triggered by liposomal fusion. The fusogenic liposome had a protein-DNA complex core containing an ATP-responsive DNA scaffold with doxorubicin (DOX) and could release DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. A cell-penetrating peptide-modified fusogenic liposomal membrane was coated on the core, which had an acid-triggered fusogenic potential with the ATP-loaded liposomes or endosomes/lysosomes. Directly delivering extrinsic liposomal ATP promoted the drug release from the fusogenic liposome in the acidic intracellular compartments upon a pH-sensitive membrane fusion and anticancer efficacy was enhanced both in vitro and in vivo. PMID:24764317

Mo, Ran; Jiang, Tianyue; Gu, Zhen



Changes in dermal interstitial ATP levels during local heating of human skin.  


Heating skin is believed to activate vanilloid type III and IV transient receptor potential ion channels (TRPV3, TRPV4, respectively), resulting in the release of ATP into the interstitial fluid. We examined the hypothesis that local skin heating would result in an accumulation of ATP in the interstitial fluid that would be related with a rise in skin blood flow (SkBF) and temperature sensation. Two microdialysis probes were inserted into the dermis on the dorsal aspect of the forearm in 15 young, healthy subjects. The probed skin was maintained at 31°C, 35°C, 39°C and 43°C for 8 min periods, during which SkBF was monitored as cutaneous vascular conductance (CVC). Dialysate was collected and analysed for ATP ([ATP](d)) using a luciferase-based assay, and ratings of perceived warmth were taken at each temperature. At a skin temperature of 31°C, [ATP](d) averaged 18.93 ± 4.06 nm and CVC averaged 12.57 ± 1.59% peak. Heating skin to 35°C resulted in an increase in CVC (17.63 ± 1.27% peak; P < 0.05), but no change in [ATP](d). Heating skin to 39°C and 43°C resulted in a decreased [ATP](d) (5.88 ± 1.68 nm and 8.75 ± 3.44 nm, respectively; P < 0.05), which was accompanied by significant elevations in CVC (38.90 ± 1.37% peak and 60.32 ± 1.95% peak, respectively; P < 0.05). Ratings of perceived warmth increased in proportion to the increase in skin temperature (r(2) = 0.75, P < 0.05). In conclusion, our data indicate that an accumulation of interstitial ATP does not occur during local heating, and therefore does not have a role in temperature sensation or the dilator response in human skin. Nevertheless, the low threshold of dilatation (35°C) indicates a possible role for the TRPV3, TRPV4 channels or the sensitization of other ion channels in mediating the dilator response. PMID:23045344

Gifford, Jayson R; Heal, Cory; Bridges, Jarom; Goldthorpe, Scott; Mack, Gary W



Diarylquinolines target subunit c of mycobacterial ATP synthase.  


The diarylquinoline R207910 (TMC207) is a promising candidate in clinical development for the treatment of tuberculosis. Though R207910-resistant mycobacteria bear mutations in ATP synthase, the compound's precise target is not known. Here we establish by genetic, biochemical and binding assays that the oligomeric subunit c (AtpE) of ATP synthase is the target of R207910. Thus targeting energy metabolism is a new, promising approach for antibacterial drug discovery. PMID:17496888

Koul, Anil; Dendouga, Najoua; Vergauwen, Karen; Molenberghs, Brenda; Vranckx, Luc; Willebrords, Rudy; Ristic, Zorica; Lill, Holger; Dorange, Ismet; Guillemont, Jerome; Bald, Dirk; Andries, Koen



Molecular characterization of the equine ATP2A2 gene  

Microsoft Academic Search

The mammalian ATP2A2 gene encodes a P-type cation pump located in the sarcoplasmic or endoplasmic reticula of muscle cells. We isolated one bacterial artificial chromosome (BAC) clone containing the equine ATP2A2 gene and determined the complete coding sequence of this gene. Cloning and characterization of the equine ATP2A2 gene revealed that the equine ATP2A2 gene consists of 20 exons. In

S. Mömke; O. Distl



Renal cell-to-cell communication via extracellular ATP.  


In the kidney, macula densa cells communicate with the mesangial cell-afferent arteriolar smooth muscle cell complex through ATP signaling. This signaling process involves release of ATP across the macula densa basolateral membrane through a maxi anion channel and the interaction of ATP with purinergic P2 receptors. PMID:15772296

Komlosi, Peter; Fintha, Attila; Bell, P Darwin



Ultrasensitive detection of ATP based on ATP regeneration amplification and its application in cell homogenate and human serum.  


A conformation-switching aptamer molecule that could be circularized without ligation DNA was designed. Pyrophosphate (PPi) was converted to ATP, resulting in higher signals for ATP detection. Meanwhile, the method has significant implications for real applications. PMID:24898261

Guo, Yingshu; Sun, Xiaofeng; Yang, Guangxu; Liu, Jia



Luminescense Determination of Cellular ATP in Cow's Milk for Mastitis Screening (Luminesnsbestamning av Cellular ATP i Komjolk for Screening Juverinflammation).  

National Technical Information Service (NTIS)

For the purpose of determining the possibility of using cellular ATP in cow's milk as a fast diagnostic test for mastitis conditions for such analysis were studied by means of the ATP-specific firefly luciferase. A simplified method of extracting ATP from...

T. Olsson K. Sandstedt O. Holmberg A. Thore



ATP-binding cassette transporters in liver.  


The human ATP-binding cassette (ABC) superfamily consists of 48 members with 14 of them identified in normal human liver at the protein level. Most of the ABC members act as ATP dependent efflux transport systems. In the liver, ABC transporters are involved in diverse physiological processes including export of cholesterol, bile salts, and metabolic endproducts. Consequently, impaired ABC transporter function is involved in inherited diseases like sitosterolemia, hyperbilirubinemia, or cholestasis. Furthermore, altered expression of some of the hepatic ABCs have been associated with primary liver tumors. This review gives a short overview about the function of hepatic ABCs. Special focus is addressed on the localization and ontogenesis of ABC transporters in the human liver. In addition, their expression pattern in primary liver tumors is discussed. PMID:24105869

Wlcek, Katrin; Stieger, Bruno



Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP.  


Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel. PMID:17656102

Jin, Jun-Yup; Park, Sung-Hee; Bae, Jae-Hoon; Cho, Ho-Chan; Lim, Jeong-Geun; Park, Won Sun; Han, Jin; Lee, Jin Ho; Song, Dae-Kyu




Microsoft Academic Search

The possible implication of P2-purinoceptors in brain functions is reviewed. Involvement of P2-purinoceptors in memory and learning (Section 2) is suggested by ATP release from hippocampal slices [Wieraszko et al. (1989)Brain Res. 485, 244–250], induction of fast synaptic currents in cultured hippocampal neurons [Inoue et al. (1992a)Neurosci. Lett. 134, 294–299] and long-lasting enhancement of the population spikes [Wieraszko and Seyfried




Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology  

NASA Technical Reports Server (NTRS)

The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

Chappelle, E. W.; Picciolo, G. L.



ATP sensitivity of ATP-sensitive K+ channels: role of the gamma phosphate group of ATP and the R50 residue of mouse Kir6.2.  


ATP-sensitive K (K(ATP)) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the alpha and beta phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the gamma or beta phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the gamma phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's gamma phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54. PMID:16166157

John, Scott A; Weiss, James N; Ribalet, Bernard



Potentiation of cytokine induction of group IIA phospholipase A2 in rat mesangial cells by ATP and adenosine via the A2A adenosine receptor  

PubMed Central

In rat mesangial cells extracellular nucleotides were found to increase arachidonic acid release by a cytosolic phospholipase A2 through the P2Y2 purinergic receptor. In this study we investigated the effects of ATP and UTP on interleukin-1? (IL-1?)-induced mRNA expression and activity of group IIA phospholipase A2 (sPLA2-IIA) in rat mesangial cells. Treatment of cells for 24?h with extracellular ATP potentiated IL-1?-stimulated sPLA2-IIA induction, whereas UTP had no effect. We obtained the following evidence that the P2Y2 receptor is not involved in the potentiation of sPLA2-IIA induction: (i) ATP-?-S had no enhancing effect; (ii) suramin, a P2 receptor antagonist, did not inhibit ATP-mediated potentiation; (iii) inhibition of degradation of extracellular nucleotides by the 5?-ectonucleotidase inhibitor AOPCP did not enhance sPLA2-IIA induction and (iv) adenosine deaminase treatment completely abolished the ATP-mediated potentiation of sPLA2-IIA induction. In contrast, treatment of mesangial cells with adenosine or the A2A receptor agonist CGS 21680 mimicked the effects of ATP in enhancing IL-1?-stimulated sPLA2-IIA induction, whereas the specific A2A receptor antagonist ZM 241385 completely abolished the potentiating effect of ATP or adenosine. The protein kinase A inhibitor Rp-8-Br-cyclic AMPS dose-dependently inhibited the enhancing effect of ATP or adenosine indicating the participation of an adenosine receptor-mediated cyclic AMP-dependent signalling pathway. These data indicate that ATP mediates proinflammatory long-term effects in rat mesangial cells via its degradation product adenosine through the A2A receptor resulting in potentiation of sPLA2-IIA induction.

Scholz-Pedretti, Kirsten; Pfeilschifter, Josef; Kaszkin, Marietta



ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer.  


ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of membrane excitability in many cell types. ATP inhibits channel activity by binding to a specific site formed by the N and C termini of the pore-forming subunit, Kir6.2, but the structural changes associated with this interaction remain unclear. Here, we use fluorescence resonance energy transfer (FRET) to study the ATP-dependent interaction between the N and C termini of Kir6.2 using a construct bearing fused cyan and yellow fluorescent proteins (ECFP-Kir6.2-EYFP). When expressed in human embryonic kidney cells, ECFP-Kir6.2-EYFP/SUR1 channels displayed FRET that was augmented by agonist stimulation and diminished by metabolic poisoning. Addition of ATP to permeabilized cells or isolated plasma membrane sheets increased FRET. FRET changes were abolished by Kir6.2 mutations that altered ATP-dependent channel closure and channel gating. In the wild-type channel, the ATP concentrations, which increased FRET (EC(50) = 1.36 mM), were significantly higher than those causing channel inhibition (IC(50) = 0.29 mM). Demonstrating the existence of intermolecular interactions, a dimeric construct comprising two molecules of Kir6.2 linked head-to-tail (ECFP-Kir6.2-Kir6.2-EYFP) displayed less FRET than the monomer in the absence of nucleotide but still exhibited ATP-dependent FRET increases (EC(50) = 1.52 mM) and channel inhibition. We conclude that binding of ATP to Kir6.2, (i). alters the interaction between the N- and C-terminal domains, (ii). probably involves both intrasubunit and intersubunit interactions, (iii). reflects ligand binding not channel gating, and (iv). occurs in intact cells when subplasmalemmal [ATP] changes in the millimolar range. PMID:14681552

Tsuboi, Takashi; Lippiat, Jonathan D; Ashcroft, Frances M; Rutter, Guy A



Sphingomyelinase Treatment Induces ATP-independent Endocytosis  

PubMed Central

ATP hydrolysis has been regarded as a general requirement for internalization processes in mammalian cells. We found, however, that treatment of ATP-depleted macrophages and fibroblasts with exogenous sphingomyelinase (SMase) rapidly induces formation of numerous vesicles that pinch off from the plasma membrane; the process is complete within 10 min after adding SMase. By electron microscopy, the SMase-induced vesicles are ?400 nm in diameter and lack discernible coats. 15–30% of plasma membrane is internalized by SMase treatment, and there is no detectable enrichment of either clathrin or caveolin in these vesicles. When ATP is restored to the cells, the SMase-induced vesicles are able to deliver fluid-phase markers to late endosomes/lysosomes and return recycling receptors, such as transferrin receptors, back to the plasma membrane. We speculate that hydrolysis of sphingomyelin on the plasma membrane causes inward curvature and subsequent fusion to form sealed vesicles. Many cell types express a SMase that can be secreted or delivered to endosomes and lysosomes. The hydrolysis of sphingomyelin by these enzymes is activated by several signaling pathways, and this may lead to formation of vesicles by the process described here.

Zha, Xiaohui; Pierini, Lynda M.; Leopold, Philip L.; Skiba, Paul J.; Tabas, Ira; Maxfield, Frederick R.




... instrument was developed by the Naval Research Laboratory (NRL). POAM III was launched aboard the French SPOT-4 satellite in March 1998 ... Relevant Documents:  POAM Information at NRL POAM II Data Table Status of Version 4 Retrievals ...



Real-time luminescence imaging of cellular ATP release.  


Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin-luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin-luciferase luminescence at ?10 frames/s, sufficient to follow rapid ATP release with sensitivity of ?10 nM and dynamic range up to 100?M. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility. PMID:23973809

Furuya, Kishio; Sokabe, Masahiro; Grygorczyk, Ryszard



ATP synthases from archaea: the beauty of a molecular motor.  


Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. PMID:24650628

Grüber, Gerhard; Manimekalai, Malathy Sony Subramanian; Mayer, Florian; Müller, Volker



Case Definition and Design Sensitivity  

PubMed Central

In a case-referent study, cases of disease are compared to non-cases with respect to their antecedent exposure to a treatment in an effort to determine whether exposure causes some cases of the disease. Because exposure is not randomly assigned in the population, as it would be if the population were a vast randomized trial, exposed and unexposed subjects may differ prior to exposure with respect to covariates that may or may not have been measured. After controlling for measured pre-exposure differences, for instance by matching, a sensitivity analysis asks about the magnitude of bias from unmeasured covariates that would need to be present to alter the conclusions of a study that presumed matching for observed covariates removes all bias. The definition of a case of disease affects sensitivity to unmeasured bias. We explore this issue using: (i) an asymptotic tool, the design sensitivity, (ii) a simulation for finite samples, and (iii) an example. Under favorable circumstances, a narrower case definition can yield an increase in the design sensitivity, and hence an increase in the power of a sensitivity analysis. Also, we discuss an adaptive method that seeks to discover the best case definition from the data at hand while controlling for multiple testing. An implementation in R is available as SensitivityCaseControl.

Small, Dylan S.; Cheng, Jing; Halloran, M. Elizabeth; Rosenbaum, Paul R.



Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase ?-Subunit as a Novel Plant Cell Death Regulator*  

PubMed Central

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase ?-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase ?-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.

Chivasa, Stephen; Tome, Daniel F. A.; Hamilton, John M.; Slabas, Antoni R.



[Structure and function of ATP7A and ATP7B proteins--Cu-transporting ATPases].  


Living organisms have developed refined and geneticaly controlled mechanisms of the copper metabolism and transport. ATP7A and ATP7B proteins play the key role in copper homeostasis in the organism. Both proteins are P-type Cu-transporting ATPases and use the energy of ATP hydrolysis to transfer the copper ions across the cellular membranes. Both proteins are localised in Golgi aparatus and involved in regulation of overall copper status in the body and their function is the export of excess copper from the cells and delivery of copper ions to Cu-dependent enzymes. Moreover in organism Cu-transporting ATPases are involved in absorption of dietary copper, Cu removal with the bile, placental copper transport and its secretion to the milk during lactation. Moreover it is known that Cu-transporting ATPases play a role in generation of anti-cancer drug resistance. Disturbances of ATP7A and ATP7B function caused by mutations lead to severe metabolic diseases Menkes and Wilson diseases, respectively. PMID:21117320

Lenartowicz, Ma?gorzata; Krzeptowski, Wojciech



Clinical definitions of melioidosis.  


Clinical definitions of melioidosis and inhalation-acquired melioidosis (Burkholderia pseudomallei infection) are described together with the evidence used to develop these definitions. Such definitions support accurate public health reporting, preparedness planning for deliberate B. pseudomallei release, design of experimental models, and categorization of naturally acquired melioidosis. PMID:23468355

Cheng, Allen C; Currie, Bart J; Dance, David A B; Funnell, Simon G P; Limmathurotsakul, Direk; Simpson, Andrew J H; Peacock, Sharon J



A New Metal-Binding Site for Yeast Phosphoglycerate Kinase as Determined by the Use of a Metal-ATP Analog  

PubMed Central

Suicide substrate ?, ?-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK). Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides. One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part, to the coordination of metal ion of the metal-ATP substrate. Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the “closed” active site. Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK·3-phospho-D-glycerate·RhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes. ImagesFIGURE 6

Pappu, Kameshwari M.; Kunnumal, Baburaj; Serpersu, Engin H.



Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection  

PubMed Central

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosis universal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-Å-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

Bilder, Patrick; Sun, Meihao; Lim, Jihyeon; Bielefeldt-Ohmann, Helle; Basaraba, Randall; So, Melvin; Zhu, Guofeng; Tufariello, JoAnn M.; Izzo, Angelo A.; Orme, Ian M.; Almo, Steve C.; Leyh, Thomas S.; Chan, John



ATP sensitivity of the ATP-sensitive K+ channel in intact and permeabilized pancreatic beta-cells.  


ATP-sensitive K(+) channels (K(ATP) channels) couple cell metabolism to electrical activity and thereby to physiological processes such as hormone secretion, muscle contraction, and neuronal activity. However, the mechanism by which metabolism regulates K(ATP) channel activity, and the channel sensitivity to inhibition by ATP in its native environment, remain controversial. Here, we used alpha-toxin to permeabilize single pancreatic beta-cells and measure K(ATP) channel ATP sensitivity. We show that the channel ATP sensitivity is approximately sevenfold lower in the permeabilized cell than in the inside-out patch and that this is caused by interaction of Mg-nucleotides with the nucleotide-binding domains of the SUR1 subunit of the channel. The ATP sensitivity observed in permeabilized cells accounts quantitatively for K(ATP) channel activity in intact cells. Thus, our results show that the principal metabolic regulators of K(ATP) channel activity are MgATP and MgADP. PMID:16936192

Tarasov, Andrei I; Girard, Christophe A J; Ashcroft, Frances M



The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells.  


The ATP- and sulphonylurea-sensitivity of the ATP-sensitive K-channel was measured in human pancreatic B cells. In inside-out patches, half-maximal inhibition of channel activity was produced by 10 mumol/l ATP (with 2 mM Mg2+) and ATP-inhibition was partially antagonised by ADP. A significantly lower sensitivity to ATP was found in whole-cell recordings. Tolbutamide inhibited whole-cell ATP-sensitive K-currents half-maximally at 18 mumol/l; the sensitivity to tolbutamide was somewhat less in the inside-out patch. Ca-activated K-channels were unaffected by tolbutamide (10 mmol/l). These results resemble those found for rodent B cells and suggest that sulphonylureas exert their therapeutic effects in Type 2 (non-insulin dependent) diabetes by inhibition of the ATP-sensitive K-channel. PMID:2673893

Ashcroft, F M; Kakei, M; Gibson, J S; Gray, D W; Sutton, R



ATP sensitivity of ATP-sensitive K+ channels: role of the ? phosphate group of ATP and the R50 residue of mouse Kir6.2  

PubMed Central

ATP-sensitive K (KATP) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the ? and ? phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the ? or ? phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the ? phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's ? phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54.

John, Scott A; Weiss, James N; Ribalet, Bernard



Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence  

SciTech Connect

The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K{sup +} and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized CdS monocrystals. Aggregates of nanospheres were bathed in imaging solution, and ATP bioluminescence was monitored to investigated the release kinetics of the nanosphere drug delivery systems. Addition of disulfide bond-cleaving molecules induced uncapping of the nanospheres and subsequently, the release of ATP. Increasing the concentration of the uncapping molecule decreased the temporal maximum and increased the magnitude of release of encapsulated ATP from the nanospheres. Furthermore, the release kinetics from the nanospheres varied with the size of the particle aggregates.

Jason Alan Gruenhagen



Coupled ATP and potassium efflux from intercalated cells  

PubMed Central

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (?; 10 dynes/cm2) -induced, BK-dependent, K efflux. We found that 10 ?M ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P2 receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited ?-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-?4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and ?-induced K efflux. Similarly, carbenoxolone (25 ?M), which blocks connexins, putative ATP pathways, blocked ?-induced K efflux and ATP secretion. Compared with BK-?4?/? mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for ?-induced ATP release in IC.

Holtzclaw, J. David; Cornelius, Ryan J.; Hatcher, Lori I.



Relation of rumen ATP concentration to bacterial and protozoal numbers.  

PubMed Central

Cultures of Streptococcus bovis and mixed populations of rumen bacteria were used to investigate the concentration of ATP and rumen bacterial numbers at various stages of growth. ATP, extracted with Tris buffer, was analyzed using the firefly luciferin-luciferase bioluminescent reaction. ATP concentrations of S. bovis and mixed cultures of rumen bacteria significantly correlated with live cell counts during the log phase of growth but not during the stationary phase. The average cellular ATP concentration of rumen bacteria was calculated to be 0.3 fg of ATP per cell. Studies done with in vivo artificial rumen apparatus revealed that the protozoal contribution to rumen fluid ATP pool size was much more substantial than was the bacterial contribution. The rumen fluid ATP concentration was greater in cattle with protozoa than in those that were defaunated. Differences in ATP concentration due to size differences of ciliate protozoa were observed. Due to the unbalanced distribution of ATP in rumen microbes, ATP appears to be an unsuitable indicator of rumen microbial biomass.

Nuzback, D E; Bartley, E E; Dennis, S M; Nagaraja, T G; Galitzer, S J; Dayton, A D



Release of extracellular ATP by bacteria during growth  

PubMed Central

Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology.



Thioredoxin-insensitive plastid ATP synthase that performs moonlighting functions  

PubMed Central

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory ? subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only ?1 is involved in ATP synthesis in photosynthesis. The ?1-containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only ?2-ATP synthase (gamma exchange-revised ATP synthase, gamera) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on ?2-ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in ?2 is substantially higher than that for ?1. This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that ?2-ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that ?2 originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses.

Kohzuma, Kaori; Dal Bosco, Cristina; Kanazawa, Atsuko; Dhingra, Amit; Nitschke, Wolfgang; Meurer, Jorg; Kramer, David M.



A genetically encoded fluorescent reporter of ATP/ADP ratio  

PubMed Central

A fluorescent sensor of adenylate nucleotides was constructed by combining a circularly permuted variant of green fluorescent protein with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The affinity for Mg-ATP is below 100 nM, as seen for the other members of the bacterial PII regulator family – a surprisingly high affinity given normal intracellular [ATP] in the millimolar range. ADP binds to the same site, competing with Mg-ATP but producing a smaller change in fluorescence. With normal physiological concentrations of ATP and ADP, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP/ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP/ADP ratio during live cell imaging.

Berg, Jim; Hung, Yin Pun; Yellen, Gary



Functional Analysis of the Streptomyces coelicolor NrdR ATP-Cone Domain: Role in Nucleotide Binding, Oligomerization, and DNA Interactions? †  

PubMed Central

Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of dNTPs during the cell cycle. Streptomyces species contain genes, nrdAB and nrdJ, coding for oxygen-dependent class I and oxygen-independent class II RNRs, either of which is sufficient for vegetative growth. Both sets of genes are transcriptionally repressed by NrdR. NrdR contains a zinc ribbon DNA-binding domain and an ATP-cone domain similar to that present in the allosteric activity site of many class I and class III RNRs. Purified NrdR contains up to 1 mol of tightly bound ATP or dATP per mol of protein and binds to tandem 16-bp sequences, termed NrdR-boxes, present in the upstream regulatory regions of bacterial RNR operons. Previously, we showed that the ATP-cone domain alone determines nucleotide binding and that an NrdR mutant defective in nucleotide binding was unable to bind to DNA probes containing NrdR-boxes. These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations.

Grinberg, Inna; Shteinberg, Tatyana; Hassan, A. Quamrul; Aharonowitz, Yair; Borovok, Ilya; Cohen, Gerald



Coupling proton movement to ATP synthesis in the chloroplast ATP synthase.  


The chloroplast F(0)F(1)-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the gamma subunit of the chloroplast F(1) enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the dark. The three-dimensional structure of the chloroplast F(1) gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial gamma subunits, together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F(0), the transmembrane proton channel portion of the enzyme, can be coupled, via the epsilon subunit, to rotation of the gamma subunit of F(1) to achieve the 120 degrees (or 90 degrees +30 degrees) stepping action that is characteristic of F(1) gamma subunit rotation. PMID:16691485

Richter, Mark L; Samra, Hardeep S; He, Feng; Giessel, Andrew J; Kuczera, Krzysztof K



Welding III.  

ERIC Educational Resources Information Center

Instructional objectives and performance requirements are outlined in this course guide for Welding III, an advanced course in arc welding offered at the Community College of Allegheny County to provide students with the proficiency necessary for industrial certification. The course objectives, which are outlined first, specify that students will…

Allegheny County Community Coll., Pittsburgh, PA.



EPA Science Inventory

LandView III is a desktop mapping system that includes database extracts from the Environmental Protection Agency, the Bureau of the Census, The U.S. Geological Survey, the Nuclear Regulatory Commission, the Department of Transportation, and the Federal Emergency Management Agenc...


Two ATP-activated conductances in bullfrog atrial cells  

Microsoft Academic Search

A B S T R A C T Currents activated by extracellular ATP were studied in single voltage-clamped bullfrog atrial cells. Rapid application of ATP elicited currents carried through two different conductance pathways: a rapidly desensitizing conductance reversing near -10 mV, and a maintained, inwardly rectifying conductance reversing near -85 mV. ATP activated the desensitizing compo- nent of current with




ATP7B detoxifies silver in ciliated airway epithelial cells  

PubMed Central

Silver is a centuries old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds, but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA treated HepG2 cells. Additionally, mTEC from ATP7B-/- mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell-type specific expression of the Ag+/Cu+ transporters ATP7A, ATP7B and CTR1 in airway epithelial cells, and a role for ATP7B in detoxification of these metals in the lung.

Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.



ATP enhances repair of hair bundles in sea anemones.  


Hair bundle mechanoreceptors of sea anemones are similar to those of the acousticolateralis system of vertebrates (Watson, Mire and Hudson, 1997, Hear. Res. 107, 53-63). Anemone hair bundles are repaired by 'repair proteins' secreted following a complete loss of structural integrity and loss of function caused by 1 h exposure to calcium free seawater. Exogenously supplied repair proteins (RP) restore structural integrity to hair bundles and restore vibration sensitivity in 7-8 min (Watson, Mire and Hudson, 1998, Hear. Res. 115, 119-128). We here report that exogenously supplied ATP enhances the rate by which RP restore vibration sensitivity. A bimodal dose response to ATP indicates maximal enhancement at picomolar and micromolar concentrations of ATP. At these concentrations of ATP, vibration sensitivity is restored in 2 min. These data suggest that at least two ATPases exhibiting different binding affinities for ATP are involved in the repair process. Whereas the higher affinity site is specific for ATP, the lower affinity site does not discriminate between ATP and ADP. Nucleotidase cytochemistry localizes ATPase activity in isolated repair proteins. In the absence of exogenously added RP, sea anemones secrete and consume ATP during the 4 h recovery period after 1 h exposure to calcium free seawater. In the presence of exogenously added RP, ATP is secreted and then consumed within 10 min. Quinacrine cytochemistry localizes possible stores of ATP in the apical cytoplasm of sensory neurons located at the center of the hair bundle. According to our model, ATP is secreted by the sensory neuron after its hair bundle loses structural integrity. Hydrolysis of ATP by repair proteins is essential to the repair process. PMID:10511619

Watson, G M; Venable, S; Hudson, R R; Repass, J J



ATP5H/KCTD2 locus is associated with Alzheimer's disease risk.  


To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12?040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10(-6) were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10(-7) in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10(-9)). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation. PMID:23857120

Boada, M; Antúnez, C; Ramírez-Lorca, R; DeStefano, A L; González-Pérez, A; Gayán, J; López-Arrieta, J; Ikram, M A; Hernández, I; Marín, J; Galán, J J; Bis, J C; Mauleón, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Morón, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Sáez, M E; Vázquez, E; Becker, J T; López, O L; Serrano-Ríos, M; Tárraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A



ATP5H/KCTD2 locus is associated with Alzheimer's disease risk  

PubMed Central

To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer's disease (AD) cases; 12?040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10?6 were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10?7 in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10?9). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation.

Boada, M; Antunez, C; Ramirez-Lorca, R; DeStefano, A L; Gonzalez-Perez, A; Gayan, J; Lopez-Arrieta, J; Ikram, M A; Hernandez, I; Marin, J; Galan, J J; Bis, J C; Mauleon, A; Rosende-Roca, M; Moreno-Rey, C; Gudnasson, V; Moron, F J; Velasco, J; Carrasco, J M; Alegret, M; Espinosa, A; Vinyes, G; Lafuente, A; Vargas, L; Fitzpatrick, A L; Launer, L J; Saez, M E; Vazquez, E; Becker, J T; Lopez, O L; Serrano-Rios, M; Tarraga, L; van Duijn, C M; Real, L M; Seshadri, S; Ruiz, A



Increased circulating levels of plasma ATP in cystic fibrosis patients.  


Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin-luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single-blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0.01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the DeltaF508 genotype had a 54% (n=33, P<0.01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0.4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease. PMID:10971545

Lader, A S; Prat, A G; Jackson, G R; Chervinsky, K L; Lapey, A; Kinane, T B; Cantiello, H F



Binding of ATP by pertussis toxin and isolated toxin subunits  

SciTech Connect

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))



Effects of organophosphorus compounds on ATP production and mitochondrial integrity in cultured cells.  


Recent studies in vivo and in vitro suggested that mitochondrial dysfunction follows exposure to organophosphorus (OP) esters. As mitochondrial ATP production is important for cellular integrity, ATP production in the presence of OP neurotoxicants was examined in a human neuronal cell line (SH-SY5Y neuroblastoma cells) and primary dorsal root ganglia (DRG) cells isolated from chick embryos and subsequently cultured to achieve maturation with axons. These cell culture systems were chosen to evaluate toxic effects on the mitochondrial respiratory chain associated with exposure to OP compounds that do and do not cause OP-induced delayed neuropathy (OPIDN), a disorder preceded by inhibition of neurotoxic esterase (NTE). Concentration- and time-response studies were done in neuroblastoma cells exposed to phenyl saligenin phosphate (PSP) and mipafox, both compounds that readily induce delayed neuropathy in hens, or paraoxon, which does not. Phenylmethylsulfonyl fluoride (PMSF) was included as a non-neuropathic inhibitor of NTE. Purified neuronal cultures from 9 day-old chick embryo DRG were treated for 12 h with 1 microM PSP, mipafox, or paraoxon. In situ evaluation of ATP production measured by bioluminescence assay demonstrated decreased ATP concentrations both in neuroblastoma cells and chick DRG neurons treated with PSP. Mipafox decreased ATP production in DRG but not in SH-SY5Y cells. This low energy state was present at several levels of the mitochondrial respiratory chain, including Complexes I, II, III, and IV, although Complex I was the most severely affected. Paraoxon and PMSF were not effective at all complexes, and, when effective, required higher concentrations than needed for PSP. Results suggest that mitochondria are an important early target for OP compounds, with exposure resulting in depletion of ATP production. The targeting of neuronal, rather than Schwann cell mitochondria in DRG following exposure to PSP and mipafox was verified by loss of the mitochondrial-specific dye, tetramethylrhodamine, in these cells. No such loss was seen in paraoxon exposed neurons isolated from DRG or in Schwann cells treated with any of the test compounds. PMID:15897155

Massicotte, C; Knight, K; Van der Schyf, C J; Jortner, B S; Ehrich, M



ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase  

NASA Technical Reports Server (NTRS)

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

Hochstein, L. I.



Two conventional PKC isoforms, ? and ?I, are involved in the ATP-induced regulation of VRAC and glutamate release in cultured astrocytes  

PubMed Central

Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate PKC involvement in regulation of astrocytic VRAC using siRNA and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 ?M ATP synergistically increased the release of excitatory amino acids, measured with non-metabolized analogue of l-glutamate, d-[3H]aspartate. Both Go6976, the selective inhibitor of Ca2+-sensitive PKC?, ?I/II, and ?, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKC? and ?I/II, reduced the effects of ATP on d-[3H]aspartate release by ~45-55%. Similar results were obtained with a mixture of siRNAs targeting rat PKC? and ?I. Surprisingly, downregulation of individual ? and ?I PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKC? and ?I.

Haskew-Layton, Renee E.; Mongin, Alexander A.



ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment  

PubMed Central

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b+/Gr-1+ cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1+ population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-?1 (TGF-?1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-?1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

Bianchi, G; Vuerich, M; Pellegatti, P; Marimpietri, D; Emionite, L; Marigo, I; Bronte, V; Di Virgilio, F; Pistoia, V; Raffaghello, L



Inland Wetland Definitions  

Microsoft Academic Search

This work is the result of a year-long study of the definitions of inland wetlands in which definitions from geology, hydrogeology, hydrology, pedology, biology, systems ecology, sociology, economics, political sciences, public health and law were considered. Of these, geology, hydrogeology, hydrology, biology, systems ecology and economics are discussed in detail in this report and used in writing a final theoretical

Michael W Lefor; William C Kennard



Common Set of Definitions.  

National Technical Information Service (NTIS)

A common set of definitions for Title XX services is found in this booklet. The service descriptors were prepared from a study conducted by Boston College Graduate School of Social Work under a grant from HEW. The definitions, program elements, and activi...



Definitions of Entomological Terms  

NSDL National Science Digital Library

A list of of morphological definitions and word roots useful to Entomology students and teachers. The list contains concise and easily understandable definitions for a number of morphological and physiological terms and specifies where on the insect these terms apply. A good reference for students in introductory entomology or insect morphology classes. Requires Adobe Acrobat Reader or equivalent software to read .pdf documents.



Intracellular ATP supports TRPV6 activity via lipid kinases and the generation of PtdIns(4,5)P2  

PubMed Central

Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in Ca2+ absorption in the intestines. Both phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and cytoplasmic ATP have been proposed to be important for maintaining TRPV6 activity. To evaluate whether PtdIns(4,5)P2 and ATP affect channel activity directly or indirectly, we have used a dual approach, examining channel activity in excised patches and planar lipid bilayers. In excised inside-out patch-clamp measurements, ATP reactivated the human TRPV6 channels after current rundown only in the presence of Mg2+. The effect of MgATP was inhibited by 3 structurally different compounds that inhibit type III phosphatidylinositol 4-kinases (PI4Ks). PtdIns(4,5)P2 also activated TRPV6 in excised patches, while its precursor PtdIns(4)P had only minimal effect. These data demonstrate that MgATP provides substrate for lipid kinases, allowing the resynthesis of PtdIns(4,5)P2. To determine whether PtdIns(4,5)P2 is a direct activator of TRPV6, we purified and reconstituted the channel protein in planar lipid bilayers. The reconstituted channel showed high activity in the presence of PtdIns(4,5)P2, while PtdIns(4)P induced only minimal activity. Our data establish PtdIns(4,5)P2 as a direct activator of TRPV6 and demonstrate that intracellular ATP regulates the channel indirectly as a substrate for type III PI4Ks.—Zakharian, E., Cao, C., Rohacs, T. Intracellular ATP supports TRPV6 activity via lipid kinases and the generation of PtdIns(4,5)P2.

Zakharian, Eleonora; Cao, Chike; Rohacs, Tibor



ATP-Driven Molecular Chaperone Machines  

PubMed Central

This review is focused on the mechanisms by which ATP binding and hydrolysis drive chaperone machines assisting protein folding and unfolding. A survey of the key, general chaperone systems Hsp70 and Hsp90, and the unfoldase Hsp100 is followed by a focus on the Hsp60 chaperonin machine which is understood in most detail. Cryo-electron microscopy analysis of the E. coli Hsp60 GroEL reveals intermediate conformations in the ATPase cycle and in substrate folding. These structures suggest a mechanism by which GroEL can forcefully unfold and then encapsulate substrates for subsequent folding in isolation from all other binding surfaces. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 846–859, 2013.

Clare, Daniel K; Saibil, Helen R



The ADP and ATP transport in mitochondria and its carrier  

Microsoft Academic Search

Different from some more specialised short reviews, here a general although not encyclopaedic survey of the function, metabolic role, structure and mechanism of the ADP\\/ATP transport in mitochondria is presented. The obvious need for an “old fashioned” review comes from the gateway role in metabolism of the ATP transfer to the cytosol from mitochondria. Amidst the labours, 40 or more

Martin Klingenberg



Extracellular ATP drives systemic inflammation, tissue damage and mortality  

PubMed Central

Systemic inflammatory response syndromes (SIRS) may be caused by both infectious and sterile insults, such as trauma, ischemia-reperfusion or burns. They are characterized by early excessive inflammatory cytokine production and the endogenous release of several toxic and damaging molecules. These are necessary to fight and resolve the cause of SIRS, but often end up progressively damaging cells and tissues, leading to life-threatening multiple organ dysfunction syndrome (MODS). As inflammasome-dependent cytokines such as interleukin-1? are critically involved in the development of MODS and death in SIRS, and ATP is an essential activator of inflammasomes in vitro, we decided to analyze the ability of ATP removal to prevent excessive tissue damage and mortality in a murine LPS-induced inflammation model. Our results indeed indicate an important pro-inflammatory role for extracellular ATP. However, the effect of ATP is not restricted to inflammasome activation at all. Removing extracellular ATP with systemic apyrase treatment not only prevented IL-1? accumulation but also the production of inflammasome-independent cytokines such as TNF and IL-10. In addition, ATP removal also prevented systemic evidence of cellular disintegration, mitochondrial damage, apoptosis, intestinal barrier disruption and even mortality. Although blocking ATP receptors with the broad-spectrum P2 purinergic receptor antagonist suramin imitated certain beneficial effects of apyrase treatment, it could not prevent morbidity or mortality at all. We conclude that removal of systemic extracellular ATP could be a valuable strategy to dampen systemic inflammatory damage and toxicity in SIRS.

Cauwels, A; Rogge, E; Vandendriessche, B; Shiva, S; Brouckaert, P



ATP11B mediates platinum resistance in ovarian cancer  

PubMed Central

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.

Moreno-Smith, Myrthala; Halder, J.B.; Meltzer, Paul S.; Gonda, Tamas A.; Mangala, Lingegowda S.; Rupaimoole, Rajesha; Lu, Chunhua; Nagaraja, Archana S.; Gharpure, Kshipra M.; Kang, Yu; Rodriguez-Aguayo, Cristian; Vivas-Mejia, Pablo E.; Zand, Behrouz; Schmandt, Rosemarie; Wang, Hua; Langley, Robert R.; Jennings, Nicholas B.; Ivan, Cristina; Coffin, Jeremy E.; Armaiz, Guillermo N.; Bottsford-Miller, Justin; Kim, Sang Bae; Halleck, Margaret S.; Hendrix, Mary J.C.; Bornman, William; Bar-Eli, Menashe; Lee, Ju-Seog; Siddik, Zahid H.; Lopez-Berestein, Gabriel; Sood, Anil K.



Electrodiffusional ATP movement through CFTR and other ABC transporters  

Microsoft Academic Search

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the superfamily of ATP-binding cassette (ABC) transporters, also known as traffic ATPases. Recent studies from our laboratory determined that various members of the ABC family of transport proteins mediate the electrodiffusional movement of the nucleotide ATP. In this report, evidence for the movement of cellular nucleotides by the ABC

H. F. Cantiello



Constitutive and agonist stimulated ATP secretion in leukocytes  

PubMed Central

Release and reception of extracellular ATP by leukocytes plays a critical role in immune responses to infection, injury and cardiovascular disease. Leukocytes of both the innate, adaptive immune and central nervous system express a repertoire of cell surface receptors for ATP (P2X and P2Y receptors) and its metabolites. ATP acts as a damage-associated molecule pattern (DAMP) released by injured or dying cells. Detection of released ATP by neighboring leukocytes initiates inflammation and wound healing. However, recent evidence from our group and others suggests ATP release by leukocytes themselves serves to regulate homeostatic mechanisms and coordinate responses to external pro-inflammatory cues. Examples include the homeostatic control of intracellular calcium and regulation of migratory guidance during chemotactic response to external cues. Though there has been some progress in elucidating ATP release mechanisms of some mammalian cells types, release conduits and coupling signal transduction machinery remain larger elusive for leukocytes. Our recent studies suggest a role for secretory lysosomes in releasing ATP in monocytes. Though poorly defined, targeting ATP release mechanisms in leukocytes have great anti-inflammatory potential.

Campwala, Hinnah; Fountain, Samuel J.



Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions.  


It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin-luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate- and stilbene-derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (approximately 390 pS). The channel was selective to anions and showed significant permeability to ATP4- (PATP/PCl approximately 0.1) and MgATP2- (PATP/PCl approximately 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels. PMID:15272030

Dutta, Amal K; Sabirov, Ravshan Z; Uramoto, Hiromi; Okada, Yasunobu



Investigation of the association between ATP2B4 and ATP5B genes with colorectal cancer.  


Colorectal cancer (CRC) develops as a multi-step process which results from gradual accumulation of mutations in proto-oncogenes, tumor suppressor, and DNA repair genes. Mortality rate of CRC is very high. Therefore, development of alternative diagnostic methods which can be used in the early diagnosis is crucial. ATP2B4 gene encodes one of the four isoforms of p-type ATPase PMCA enzyme and bears critical importance in maintaining the balance of intracellular calcium homeostasis by providing the export of calcium ions out of the cell. ATP5B encodes a subunit of the mitochondrial ATP synthase which is an f-type ATPase. In this study, the relationship between ATP2B4 and ATP5B genes and CRC regarding gene expression was investigated. Study groups were constructed from a number of 50 patients (25 males, 25 females) with the mean age of 55.68±9.4 and the gene expression levels in the healthy and cancerous tissues of the patients were compared by using semi-quantitative PCR and Real-Time PCR methods. As a result, in patients with rectum tumors, there was a significant relationship between ATP2B4 gene expression and the tumor location and in patients younger than 45years, ATP5B gene expressions were detected significantly higher in tumor tissues by using RT-PCR. However, no significant relationship was detected in terms of expression differences of ATP2B4 and ATP5B genes between cancerous and healthy tissues of the CRC patients. ATP2B4 and ATP5B genes might have indirect associations in CRC pathogenesis and the investigation of their interactions with DNA repair and other related genes may help in understanding of CRC formation. PMID:24583174

Geyik, Esra; Igci, Yusuf Ziya; Pala, Elif; Suner, Ali; Borazan, Ersin; Bozgeyik, Ibrahim; Bayraktar, Emine; Bayraktar, Recep; Ergun, Sercan; Cakmak, Ecir Ali; Gokalp, Avni; Arslan, Ahmet



Mechanism of ATP loss in nonoxidative contracting muscle  

NSDL National Science Digital Library

The transition from rest to intense exercise is a challenge to cellular energetics (11, 13, 15). The metabolic fuels, i.e., the sources of ATP to sustain muscular contraction, are creatine phosphate and glycogen. Two anaerobic metabolic paths, leading to ATP generation, are catalyzed by creatine kinase and by the 12 enzymes of nonoxidative glycolysis, starting from glycogen. There is now general agreement that, unless replenished, creatine phosphate can sustain heavy muscle contraction for only 3ÃÂ4 s. Thereafter, nonoxidative glycolysis becomes the main ATP source, until the onset of fatigue. This article aimed to relate the path of ATP generation during glycogen utilization as a metabolic fuel with that of ATP breakdown in nonoxidative contracting muscle.



Irreversible desensitization of ATP responses in developing chick skeletal muscle.  

PubMed Central

1. In developing chick skeletal muscle, extracellular adenosine 5'-triphosphate (ATP) elicits an early excitatory conductance increase followed by a late potassium conductance increase. Both of these responses desensitize profoundly. Intracellular recordings and whole-cell voltage-clamp recordings were made in order to examine the time course and mechanism of desensitization and the recovery from desensitization. 2. Most of the loss of responsiveness to ATP occurred during the first minute of exposure to ATP. For the excitatory conductance, the loss of responsiveness to ATP resulted in part from long-lasting activation of the ATP-sensitive channels and in part from entrance into an inactive (non-conducting) state. In contrast, desensitization of the potassium conductance was entirely the result of a relatively fast transition to an inactive state. 3. Recovery from desensitization took many hours for both responses and was quite sensitive to temperature. 4. Recovery from desensitization for both responses was prevented by preincubation with the glycosylation inhibitor, tunicamycin. Several lines of evidence suggest that tunicamycin treatment blocked the delivery of new ATP receptors to the cell surface. 5. The recovery of the early response to ATP following exposure to two non-competitive inhibitors of the ATP response was also examined. These two compounds are thought to covalently modify the receptor. After exposure to either of these inhibitors, responsiveness to ATP returned over a time course that was similar to the time course of recovery from desensitization. 6. These results indicate that, following activation, ATP receptors do not become available for reactivation, and that recovery from desensitization is due to the insertion of newly synthesized receptors into the plasma membrane.

Thomas, S A; Hume, R I



Regulation of ClC-2 gating by intracellular ATP.  


ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions. PMID:23632988

Stölting, Gabriel; Teodorescu, Georgeta; Begemann, Birgit; Schubert, Julian; Nabbout, Rima; Toliat, Mohammad Reza; Sander, Thomas; Nürnberg, Peter; Lerche, Holger; Fahlke, Christoph



Involvement of connexin43 hemichannel in ATP release after ?-irradiation.  


Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that ?-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X(7) receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X(7) receptor-dependent ATP release after 0.5 Gy ?-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked ?-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy ?-irradiation tyrosine phosphorylation was significantly blocked by P2X(7) receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X(7) receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X(7) receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X(7) receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43. PMID:22843620

Ohshima, Yasuhiro; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Kojima, Shuji



Reinterpreting the Action of ATP Analogs on KATP Channels*  

PubMed Central

Neuroendocrine-type KATP channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K+, and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting ?-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5?-(?,?-imino)triphosphate (AMP-PNP) and adenosine 5?-O-(thiotriphosphate) (ATP?S) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR1E1507Q, with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATP?S, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5?-(?,?-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[32P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate KATP channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Ortiz, David; Gossack, Lindsay; Quast, Ulrich; Bryan, Joseph



Phosphate and R2D2 Restrict the Substrate Specificity of Dicer-2, an ATP-Driven Ribonuclease  

PubMed Central

SUMMARY Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from pre-microRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage, and an N-terminal helicase motif whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.

Cenik, Elif Sarinay; Fukunaga, Ryuya; Lu, Gang; Dutcher, Robert; Wang, Yeming; Tanaka Hall, Traci M.; Zamore, Phillip D.



Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.  


The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment. PMID:24561583

Zimmer-Faust, Amity G; Thulsiraj, Vanessa; Ferguson, Donna; Jay, Jennifer A



Solution structures of the actuator domain of ATP7A and ATP7B, the Menkes and Wilson disease proteins.  


ATP7A and ATP7B are two human P(1B)-type ATPases that have a crucial role in maintaining copper(I) homeostasis. Among the various domains of these enzymes, one, called the Actuator or A-domain, has a regulatory function and is required for the phosphatase step of the catalytic cycle (dephosphorylation of the intermediate formed during ATP hydrolysis). Here we report the solution structures of the A-domain of both proteins, solved by heteronuclear NMR spectroscopy and a characterization of the dynamics of the A-domain of ATP7A. We observed that the catalytically important TGE loop protrudes from the structure ready for interaction with the phosphorylated site in the ATP-binding domain. The loop is rigid, suggesting that the catalytic step does not require substantial structural flexibility or rearrangements. The present structures were useful to rationalize the molecular effects of disease-causing mutations. In particular, it can be concluded that mutations occurring in the A-domain either destabilize the fold of the domain (such as Gly860Val in ATP7A) or affect the network of communication within the domain (such as Leu873Arg in ATP7A) or with the other domains of the enzyme (such as Gly853Arg in ATP7A). PMID:19645496

Banci, Lucia; Bertini, Ivano; Cantini, Francesca; Migliardi, Manuele; Natile, Giovanni; Nushi, Fiorentin; Rosato, Antonio



Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue  

PubMed Central

Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.



Modulation of ATP/ADP concentration at the endothelial surface by shear stress: effect of flow-induced ATP release.  


The adenine nucleotides ATP and ADP induce the production of vasoactive compounds in vascular endothelial cells (ECs). Therefore, knowledge of how flow affects the concentration of ATP and ADP at the EC surface may be important for understanding shear stress-mediated vasoregulation. The concentration of ATP and ADP is determined by convective and diffusive transport as well as by hydrolysis of these nucleotides by ectonucleotidases at the EC surface. Previous mathematical modeling has demonstrated that for steady flow in a parallel plate flow chamber, the combined ATP+ADP concentration does not change considerably over a wide range of shear stress. This finding has been used to argue that the effect of flow on adenine nucleotide transport could not account for the dependence of endothelial responses to ATP on the magnitude of applied shear stress. The present study extends the previous modeling to include pulsatile flow as well as flow-induced endothelial ATP release. Our results demonstrate that flow-induced ATP release has a pronounced effect on nucleotide concentration under both steady and pulsatile flow conditions. While the combined ATP+ADP concentration at the EC surface in the absence of flow-induced ATP release changes by only approximately 10% over the wall shear stress range 0.1-10 dyne cm(-2), inclusion of this release leads to a concentration change of approximately 34%-106% over the same shear stress range, depending on how ATP release is modeled. These results suggest that the dependence of various endothelial responses to shear stress on the magnitude of the applied shear stress may be partially attributable to flow-induced changes in cell-surface adenine nucleotide concentration. PMID:11599582

John, K; Barakat, A I



Human Services Inventory: Definitions.  

National Technical Information Service (NTIS)

Definitions are presented of 24 categories of human services and of specific programs within the categories. The service categories defined are as follows: alcoholism, children and youth, community care, community groups/associations, consumer services, c...

M. Marks



Definition of the Mesoscale  

NSDL National Science Digital Library

This module provides a working definition of mesoscale meteorology. The module briefly touches on many topics crucial to forecasting mesoscale weather phenomena, such as non-hydrostatic processes, the importance of terrain, NWP model resolution, and impact on sensible weather.

Spangler, Tim



Definition of Death.  

National Technical Information Service (NTIS)

The Legislative Service Commission on April 27, 1976, directed the investigation of the various legal definitions of death and conducted a comprehensive survey of the legal, medical, and ethical aspects of the artificial sustaining of life. This report ha...



Definition of UTC.  

National Technical Information Service (NTIS)

The current definition of Coordinated Universal Time (UTC) is related to the unpredictable, variable rotation rate of the Earth. This is accomplished by irregular insertions of leap seconds, which create unpredictable discontinuities in the UTC. With the ...

D. D. McCarthy



Synthesis and fluorescence characteristics of ATP-based FRET probes.  


Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2'- and the ?-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the ?- and the C2- or the O3'-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes. PMID:24173528

Hardt, Norman; Hacker, Stephan M; Marx, Andreas



Dimers of mitochondrial ATP synthase form the permeability transition pore.  


Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca(2+) like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca(2+). Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca(2+), addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg(2+)/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D; Petronilli, Valeria; Zoratti, Mario; Szabó, Ildikó; Lippe, Giovanna; Bernardi, Paolo



Oxidized ATP inhibits T-cell-mediated autoimmunity.  


T cells directed against self antigens play an important role in several autoimmune diseases. The available immunosuppressive compounds used to treat autoimmune diseases are limited, and often they have side effects that limit their application. T cells express ATP receptors, which could be new target molecules to treat autoimmune disease. Here we analyzed the effect of oxidized ATP (oxATP), an inhibitor of the ATP receptor P2rx7, in different murine models of T-cell-mediated autoimmune diseases. Treatment with oxATP inhibited proliferation and effector function of T cells. In the systems we used, oxATP did not obviously interfere with the innate immune response, but strongly reduced antigen-specific T-cell responses. This treatment ameliorated T-cell-mediated autoimmune type I diabetes and autoimmune encephalitis in mice. In conclusion, oxATP was found to strongly inhibit activated T cells and could thus be used to target T-cell-mediated autoimmune disease. PMID:20683833

Lang, Philipp A; Merkler, Doron; Funkner, Pauline; Shaabani, Namir; Meryk, Andreas; Krings, Caroline; Barthuber, Carmen; Recher, Mike; Brück, Wolfgang; Häussinger, Dieter; Ohashi, Pamela S; Lang, Karl S



ATP release and purinergic signaling in NLRP3 inflammasome activation  

PubMed Central

The NLRP3 inflammasome is a protein complex involved in IL-1? and IL-18 processing that senses pathogen- and danger-associated molecular patterns (PAMPs and DAMPs). One step- or two step-models have been proposed to explain the tight regulation of IL-1? production during inflammation. Moreover, cellular stimulation triggers adenosine triphosphate (ATP) release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1? through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key pro-inflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

Gombault, Aurelie; Baron, Ludivine; Couillin, Isabelle



49 CFR 173.132 - Class 6, Division 6.1-Definitions.  

Code of Federal Regulations, 2013 CFR

...FOR SHIPMENTS AND PACKAGINGS Definitions Classification, Packing Group Assignments and Exceptions for Hazardous Materials Other...iii). (c) For purposes of classifying and assigning packing groups to mixtures possessing oral or dermal toxicity...



Building Consistent Dictionary Definitions  

Microsoft Academic Search

This paper is focused on the syntactic and semantic structures of dictionary definitions (in Czech). The former are explored\\u000a by means of the partial syntactic parser Dis and their main types are presented together with their frequencies obtained from\\u000a the sample of thousands dictionary definitions. It is shown that it is important to know valency frames for nouns that serve

Karel Pala; Eva Mráková



Chromatography Nomenclature and Definitions  

NSDL National Science Digital Library

This website gives the International Union of Pure and Applied Chemistry approved definitions in the field of chromatography. It is critical for students to appreciate the importance of using standardized nomenclature and definitions. Sections of the site include general terminology, terms related to the chromatographic system, terms related to the chromatographic process and the theory of chromatography, terms related to detection, ion exchange, liquid-liquid distribution (solvent extraction) and other related subjects.



Inhibition of TRPC5 channels by intracellular ATP.  


TRPC5 channels are Ca(2+)-permeable nonselective cation channels activated by G-protein-coupled receptors, although the mechanisms responsible for channel activation and regulation are poorly understood. Carbachol-activated TRPC5 currents were recorded by the whole-cell patch clamp technique from human embryonic kidney 293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. Some published studies of TRPC5 currents have included ATP and/or GTP in the patch pipette, whereas others used an ATP- and GTP-free pipette solution. We initially included these two nucleotides in the patch pipette but found that TRPC5 currents were absent or were very small. Recordings made with an ATP- and GTP-free pipette solution produced large and robust TRPC5 currents. Under these conditions, treatment of cells with Pasteurella multocida toxin, a selective inhibitor of Galpha(q), almost abolished TRPC5 currents indicating that Galpha(q) is necessary for activation of TRPC5 by the M1 receptor. To study the effect of intracellular ATP on TRPC5 channels, an intracellular perfusion system was used. Perfusion of ADP or control pipette solution had no effect, whereas perfusion of ATP or AMP-PNP, a nonhydrolyzable analog of ATP, significantly inhibited TRPC5 currents. Thus, the effects of ATP have structural specificity and probably involve a direct effect on the channel rather than a phosphorylation-mediated effect. The activity of TRPC5 channels may be linked to cellular metabolism via changes in ATP levels and could be involved in Ca(2+) overload occurring after ischemia when ATP is depleted. PMID:17925457

Dattilo, Michael; Penington, Nicholas J; Williams, Keith



Modeling the ATP production in mitochondria.  


We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer's model. We identify some inaccuracies in the BPLS original approximations for two flux rates, namely the adenine nucleotide translocator rate JANT and the calcium uniporter rate Juni. We introduce new approximations for such flux rates and then analyze some of the dynamical properties of the model. We infer, from exhaustive numerical explorations, that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs, as one would indeed expect from homeostasis. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium Cac and the substrate fructose 1,6-bisphosphate FBP. The same dynamical effects of calcium and FBP saturations reported for the original BPLS model are observed here. We find out, however, a novel nonstationary effect, which could be, in principle, physiologically interesting: some response times of the model tend to increase considerably for high concentrations of calcium and/or FBP. In particular, the larger the concentrations of Cac and/or FBP, the larger the necessary time to attain homeostasis. PMID:23760661

Saa, Alberto; Siqueira, Kellen M



Preparation and configurational analysis of the stereoisomers of /beta/,/gamma/-bidentate Rh(H/sub 2/O)/sub 4/ATP and /alpha/,/beta/,/gamma/-tridentate Rh(H/sub 2/O)/sub 3/ATP. A new class of enzyme active site probes  

SciTech Connect

Exchange-inert Co(III) and Cr(III) complexes of polyphosphates have proved to be useful probes of the structural and biochemical properties of naturally occurring Mg/sup II/(polyphosphate) complexes. However, applications of these complexes are not without limitations. The Cr/sup III/(polyphosphate) probes or their enzymatic products cannot be used in NMR methods because of the paramagnetic nature of the Cr(III) metal. The redox properties of the metal in the Co/sup III/(polyphosphate) complexes require that they also be coordinated to a nitrogen-containing ligand. This requirement is not always convenient. This work reported herein was undertaken to create a new class of exchange-inert metal polyphosphate complexes that contain a metal that is both diamagnetic and redox stable. The preparation, properties, and configurational analysis of the stereoisomers of /beta/, /gamma/-bidentate Rh(H/sub 2/O)/sub 4/ATP (ATP = adenosine 5'-triphosphate) and /alpha/,/beta/,/gamma/-tridentate Rh(H/sub 2/O)/sub 3/ATP are described. 12 refs., 5 figs.

Lu, Z.; Shorter, A.L.; Lin, I.; Dunaway-Mariano, D.



Twisting and subunit rotation in single FOF1-ATP synthase  

PubMed Central

FOF1-ATP synthases are ubiquitous proton- or ion-powered membrane enzymes providing ATP for all kinds of cellular processes. The mechanochemistry of catalysis is driven by two rotary nanomotors coupled within the enzyme. Their different step sizes have been observed by single-molecule microscopy including videomicroscopy of fluctuating nanobeads attached to single enzymes and single-molecule Förster resonance energy transfer. Here we review recent developments of approaches to monitor the step size of subunit rotation and the transient elastic energy storage mechanism in single FOF1-ATP synthases.

Sielaff, Hendrik; Borsch, Michael




PubMed Central

Akagi, J. M. (University of Kansas, Lawrence) and L. Leon Campbell. Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84:1194–1201. 1962.—Adenosine triphosphate (ATP)-sulfurylase, which catalyzes the formation of adenosine-5?-phosphosulfate (APS) from ATP and SO4=, has been purified from crude extracts of Clostridium nigrificans and Desulfovibrio desulfuricans by (NH4)2SO4 fractionation and triethylaminoethyl column chromatography. The enzyme from both sources operates over a broad pH range from 6.0 to 9.5. Below pH 6.0, activity decreases sharply, with no detectable activity at pH 5.0. Of the nucleotides tested (ATP and the triphosphates of deoxyadenosine, uridine, inosine, and guanosine), only ATP was acted upon by the enzyme from either source. The enzyme requires Mg++ for activity. Incubation of the enzyme from both organisms with ATP and S35O4= in the presence of helium resulted in the formation of an S35-labeled nucleotide whose electrophoretic mobility was identical to that of chemically prepared APS. When incubated with ATP and the group VI anions (CrO4, MoO4, WO4), the enzyme from both organisms formed an unstable intermediate, resulting in the accumulation of pyrophosphate. Thermal stability studies revealed that the ATP-sulfurylase of C. nigrificans was stable at higher temperatures than the enzyme obtained from D. desulfuricans. Exposure of the enzyme from C. nigrificans to 65 C for 2 hr gave virtually no decrease in activity. In contrast, the enzyme from D. desulfuricans was completely inactivated after 30 min at 55 C, after 3 min at 60 C, or after 1 min at 65 C.

Akagi, J. M.; Campbell, L. Leon



Lanthanide(III) complexes of bis-semicarbazone and bis-imine-substituted phenanthroline ligands: solid-state structures, photophysical properties, and anion sensing.  


Phenanthroline-based hexadentate ligands L(1) and L(2) bearing two achiral semicarbazone or two chiral imine moieties as well as the respective mononuclear complexes incorporating various lanthanide ions, such as La(III), Eu(III), Tb(III), Lu(III), and Y(III) metal ions, were synthesized, and the crystal structures of [ML(1)Cl(3)] (M=La(III), Eu(III), Tb(III), Lu(III), or Y(III)) complexes were determined. Solvent or water molecules act as coligands for the rare-earth metals in addition to halide anions. The big Ln(III) ion exhibits a coordination number (CN) of 10, whereas the corresponding Eu(III), Tb(III), Lu(III), and Y(III) centers with smaller ionic radii show CN=9. Complexes of L(2), namely [ML(2)Cl(3)] (M=Eu(III), Tb(III), Lu(III), or Y(III)) ions could also be prepared. Only the complex of Eu(III) showed red luminescence, whereas all the others were nonluminescent. The emission properties of the Eu derivative can be applied as a photophysical signal for sensing various anions. The addition of phosphate anions leads to a unique change in the luminescence behavior. As a case study, the quenching behavior of adenosine-5'-triphosphate (ATP) was investigated at physiological pH value in an aqueous solvent. A specificity of the sensor for ATP relative to adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) was found. (31)P NMR spectroscopic studies revealed the formation of a [EuL(2)(ATP)] coordination species. PMID:23150237

Nadella, Sandeep; Selvakumar, Paulraj M; Suresh, Eringathodi; Subramanian, Palani S; Albrecht, Markus; Giese, Michael; Fröhlich, Roland



Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.  


We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. PMID:24743022

da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis



Astrocytes Protect Neurons against Methylmercury via ATP/P2Y1 Receptor-Mediated Pathways in Astrocytes  

PubMed Central

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

Shibata, Keisuke; Imura, Yoshio; Morizawa, Yosuke; Gachet, Christian; Koizumi, Schuichi



Impact of Modulators of Mitochondrial ATP-Sensitive Potassium Channel (mitoK ATP ) on Hypoxic Pulmonary Vasoconstriction  

Microsoft Academic Search

Previously, we demonstrated that hypoxic pulmonary vasoconstriction (HPV) of intra-acinar arteries (IAA) requires mitochondrial\\u000a complex II (= succinate dehydrogenase, SDH) activity (citeauthor ch41:paddenberg2006, Respir Res, 7:93, citeyear ch41:paddenberg2006).\\u000a Interestingly, SDH subunits A and B have recently been described as components of a multiprotein mitochondrial ATP-sensitive\\u000a potassium channel (mitoKATP), together with mitochondrial ATP-binding cassette protein-1, adenine nucleotide translocator (ANT), ATP synthase,

R. Paddenberg; P. Faulhammer; A. Goldenberg; B. Gries; J. Heinl; W. Kummer


Synthesis and in vitro microbial evaluation of La(III), Ce(III), Sm(III) and Y(III) metal complexes of vitamin B6 drug  

NASA Astrophysics Data System (ADS)

Metal complexes of pyridoxine mono hydrochloride (vitamin B6) are prepared using La(III), Ce(III), Sm(III) and Y(III). The resulting complexes are investigated. Some physical properties, conductivity, analytical data and the composition of the four pyridoxine complexes are discussed. The elemental analysis shows that the formed complexes of La(III), Ce(III), Sm(III) and Y(III) with pyridoxine are of 1:2 (metal:PN) molar ratio. All the synthesized complexes are brown in color and possess high melting points. These complexes are partially soluble in hot methanol, dimethylsulfoxide and dimethylformamide and insoluble in water and some other organic solvents. Elemental analysis data, spectroscopic (IR, UV-vis. and florescence), effective magnetic moment in Bohr magnetons and the proton NMR suggest the structures. However, definite particle size is determined by invoking the X-ray powder diffraction and scanning electron microscopy data. The results obtained suggested that pyridoxine reacted with metal ions as a bidentate ligand through its phenolate oxygen and the oxygen of the adjacent group at the 4?-position. The molar conductance measurements proved that the pyridoxine complexes are electrolytic in nature. The kinetic and thermodynamic parameters such as: Ea, ?H*, ?S* and ?G* were estimated from the DTG curves. The antibacterial evaluation of the pyridoxine and their complexes were also performed against some gram positive, negative bacteria as well as fungi.

Refat, Moamen S.; Al-Azab, Fathi M.; Al-Maydama, Hussein M. A.; Amin, Ragab R.; Jamil, Yasmin M. S.



ATP-dependent Ca2+ uptake into plant membrane vesicles  

PubMed Central

Membrane vesicles were extracted from etiolated and light-grown plants, a plant cell suspension culture, and an alga. Upon addition of ATP and Mg2+, active Ca2+ uptake into the vesicles against a concentration gradient was shown. The dependence of this uptake on ATP and Mg2+ concentrations, pH, and temperature is described. In the absence of oxalate, equilibrium between Ca2+ uptake and efflux was reached after about 30 min. In the presence of oxalate, Ca2+ accumulation continued for at least 120 min. Ca2+ efflux from preloaded vesicles did not depend on ATP. Addition of the ionophores A23187 and Ro 20-0006/006 caused an immediate release of accumulated free Ca2+. ATP-dependent Ca2+ uptake was not inhibited by 10 ?M oligomycin.

Gross, Joachim; Marme, Dieter



Adenosine triphosphate (ATP) in the marine environment: a bibliography  

SciTech Connect

This bibliography lists published works on adenosine triphosphate (ATP) detected in the marine environment. Over 100 citations are listed in four categories: field measurements; macroscopic organisms; benthic populations; and papers on analytical methods.

Jones, A.T.; Hartwig, E.O.; Quinby-Hunt, M.S.



Affinity labeling of Avena phytochrome with ATP analogs  

PubMed Central

The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5?-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5?-triphosphate, were used to affinity label purified Avena phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys75,Ala25) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome. Images

Wong, Yum-Shing; Lagarias, J. Clark



Distinct neurological disorders with ATP1A3 mutations.  


Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the ?3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in-vitro and animal model systems, and the role of Na(+)/K(+)-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

Heinzen, Erin L; Arzimanoglou, Alexis; Brashear, Allison; Clapcote, Steven J; Gurrieri, Fiorella; Goldstein, David B; Jóhannesson, Sigurður H; Mikati, Mohamad A; Neville, Brian; Nicole, Sophie; Ozelius, Laurie J; Poulsen, Hanne; Schyns, Tsveta; Sweadner, Kathleen J; van den Maagdenberg, Arn; Vilsen, Bente



ATP Production in Chlamydomonas reinhardtii Flagella by Glycolytic Enzymes  

PubMed Central

Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half of glycolysis (phosphoglycerate mutase, enolase, and pyruvate kinase). These enzymes can generate one ATP molecule for every substrate molecule consumed. Flagellar fractionation shows that enolase is at least partially associated with the axoneme, whereas phosphoglycerate mutase and pyruvate kinase primarily reside in the detergent-soluble (membrane + matrix) compartments. We further show that axonemal enolase is a subunit of the CPC1 central pair complex and that reduced flagellar enolase levels in the cpc1 mutant correlate with the reduced flagellar ATP concentrations and reduced in vivo beat frequencies reported previously in the cpc1 strain. We conclude that in situ ATP synthesis throughout the flagellar compartment is essential for normal flagellar motility.

Mitchell, Beth F.; Pedersen, Lotte B.; Feely, Michael; Rosenbaum, Joel L.; Mitchell, David R.



ATP modulation of calcium channels in chromaffin cells.  

PubMed Central

1. The effects of externally applied micromolar concentrations of adenosine 5'-triphosphate (ATP) on Ca2+ currents (ICa) were studied in whole-cell clamped adrenaline-secreting chromaffin cells. 2. Ca2+ currents in chromaffin cells activated at about -40 mV, reached a maximum at 0 mV and had an apparent reversal potential at +50 to +60 mV, indicating the existence of only high voltage-activated Ca2+ channels. 3. ATP blocked Ca2+ current rapidly, reversibly and in a concentration-dependent manner (10(-9)-10(-4) M). 4. ATP did not completely block Ca2+ current even at the highest concentrations used (100 microM). The remaining component of Ca2+ current was characterized by slower activation and inactivation kinetics. 5. ATP blocked ICa even in the presence of nisoldipine and/or omega-conotoxin GVIA, suggesting that its modulatory role is not specific for L- and/or N-type Ca2+ channels. 6. Other adenine nucleotides also blocked the Ca2+ current partially. The order of potencies was ATP > or = ADP > AMP >> adenosine, indicating that the ATP effects are most probably mediated by a P2-type purinergic receptor. 7. Dialysis of the cells with an intracellular solution containing 1 mM guanosine 5'-O-thiodiphosphate (GDP-beta-S) or pre-incubation of the cells with pertussis toxin (PTX) blocked the inhibitory effects of ATP. 8. Intracellular application of the non-hydrolysable GTP analogue guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma-S; 50 microM) also decreased ICa in a manner similar to that seen for ATP and significantly reduced the ATP inhibitory effect. 9. Conditioning pulses to potentials positive to +80 mV partly reversed the inhibitory effects of ATP on the Ca2+ current. The prepulse-induced enhancement of ICa depended on [GTP]i-related G protein activity such that concentrations larger than 200 microM GTP, or GTP-gamma-S (50 microM) were required for significant prepulse potentiation of the Ca2+ current, while dialysis with GDP-beta-S prevented it. 10. We conclude that the ATP, co-released with catecholamines in the intact adrenal gland, may inhibit the secretory process by down-regulating the Ca2+ channel via a P2-type purinergic receptor coupled to a PTX-sensitive G protein.

Gandia, L; Garcia, A G; Morad, M



Ecotourism: The Evolving Contemporary Definition  

Microsoft Academic Search

A rise in the popularity of ecotourism has coincided with voluminous definitional discourse. Amongst stakeholders, confusion has resulted from the disparate nature of these definitions. In the absence of a common definition or set of key tenets the challenge has been to ensure operational ecotourism that adheres to the theoretical underpinnings of the concept. Without some semblance of definitional consensus,

Holly M. Donohoe; Roger D. Needham



In vitro antimycobacterial spectrum of a diarylquinoline ATP synthase inhibitor.  


The diarylquinoline R207910 is in clinical development for tuberculosis treatment. The MIC(50) for 41 drug-susceptible and 44 multidrug-resistant Mycobacterium tuberculosis clinical isolates was 0.032 microg/ml. Out of 20 additional mycobacterial species, three were found to be naturally resistant to R207910 and were shown to exhibit a polymorphism in their atpE genes. PMID:17709466

Huitric, Emma; Verhasselt, Peter; Andries, Koen; Hoffner, Sven E



Improved methodology for ATP determination in marine environments  

Microsoft Academic Search

A simple technique is described for de-salting and concentrating adenosine triphosphate (ATP) extracted from seawater or marine sediment samples prior to assays with the standard luciferin-luciferase procedure. The technique involves chromatography of H2SO4 extracts on columns of activated carbon. The efficiency of ATP recovery from marine sediments using this pre-treatment was superior to that attained with either boiling “Tris” extraction

R. E. Hodson; O. Holm-Hansenand; F. Azam



ATP can be dispensable for prespliceosome formation in yeast  

Microsoft Academic Search

The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway,

Rhonda Perriman; Manuel Ares


An aptamer-based electrochemiluminescent biosensor for ATP detection  

Microsoft Academic Search

An aptamer-based electrochemiluminescent (ECL-AB) biosensor for ATP detection with high sensitivity and specificity was developed. The biosensor was assembled based on several steps. First, a complementary DNA (cDNA) of the ATP-binding aptamer, which has six complementary bases at both its ends, was hybridized with the aptamer molecule to form a rigid, linear double-stranded DNA (ds-DNA). The ds-DNA was then labeled

Wu Yao; Lun Wang; Haiyan Wang; Xiaolei Zhang; Ling Li



Inhibition of ATP-sensitive potassium channels by haloperidol.  


Chronic haloperidol treatment has been associated with an increased incidence of glucose intolerance and type-II diabetes mellitus. We studied the effects of haloperidol on native ATP-sensitive potassium (K(ATP)) channels in mouse pancreatic beta cells and on cloned Kir6.2/SUR1 channels expressed in HEK293 cells. The inhibitory effect of haloperidol on the K(ATP) channel was not mediated via the D2 receptor signaling pathway, as both D2 agonists and antagonists blocked the channel. K(ATP) currents were studied using the patch-clamp technique in whole-cell and outside-out patch configurations. Addition of haloperidol to the extracellular solution inhibited the K(ATP) conductance immediately, in a reversible and voltage-independent manner. Haloperidol did not block the channel when applied intracellularly in whole-cell recordings. Haloperidol blocked cloned Kir6.2/SUR1 and Kir6.2DeltaC36 K(ATP) channels expressed in HEK cells. This suggests that the drug interacts with the Kir6.2 subunit of the channel. The IC(50) for inhibition of the K(ATP) current by haloperidol was 1.6 microM in 2 mM extracellular K(+) concentration ([K(+)](o)) and increased to 23.9 microM in 150 mM [K(+)](o). The Hill coefficient was close to unity, suggesting that the binding of a single molecule of haloperidol is sufficient to close the channel. Haloperidol block of K(ATP) channels may contribute to the side effects of this drug when used therapeutically. PMID:15533888

Yang, Shi-Bing; Proks, Peter; Ashcroft, Frances M; Rupnik, Marjan



ATP-regulated K+ channels in cardiac muscle  

Microsoft Academic Search

An outward current of unknown nature increases significantly when cardiac cells are treated with cyanide or subjected to hypoxia1-4, and decreases on intracellular injection of ATP5. We report here that application of the patch-clamp technique to CN-treated mammalian heart cells reveals specific K+ channels which are depressed by intracellular ATP (ATPi) at levels greater than 1 mM. For these channels,

A. Noma



Regulation of the Export of Platinum-Containing Drugs by the Copper Efflux Transporters ATP7A and ATP7B  

Microsoft Academic Search

\\u000a ATP7A and ATP7B are P-type ATPases that detoxifie copper (Cu) through sequestration into the secretory pathway. While ATP7A\\u000a is ubiquitously expressed, the expression of ATP7B is mainly specific to the liver tissue. These transporters are up-regulated\\u000a in many types of tumors refractory, to the platinum-(Pt) containing drugs. Studies on cell lines indicate that ATP7A and ATP7B\\u000a mediate sequestration and efflux

Roohangiz Safaei; Stephen B. Howell


Concerted gating mechanism underlying KATP channel inhibition by ATP.  


KATP channels assemble from four regulatory SUR1 and four pore-forming Kir6.2 subunits. At the single-channel current level, ATP-dependent gating transitions between the active burst and the inactive interburst conformations underlie inhibition of the KATP channel by intracellular ATP. Previously, we identified a slow gating mutation, T171A in the Kir6.2 subunit, which dramatically reduces rates of burst to interburst transitions in Kir6.2DeltaC26 channels without SUR1 in the absence of ATP. Here, we constructed all possible mutations at position 171 in Kir6.2DeltaC26 channels without SUR1. Only four substitutions, 171A, 171F, 171H, and 171S, gave rise to functional channels, each increasing Ki,ATP for ATP inhibition by >55-fold and slowing gating to the interburst by >35-fold. Moreover, we investigated the role of individual Kir6.2 subunits in the gating by comparing burst to interburst transition rates of channels constructed from different combinations of slow 171A and fast T171 "wild-type" subunits. The relationship between gating transition rate and number of slow subunits is exponential, which excludes independent gating models where any one subunit is sufficient for inhibition gating. Rather, our results support mechanisms where four ATP sites independently can control a single gate formed by the concerted action of all four Kir6.2 subunit inner helices of the KATP channel. PMID:15041650

Drain, Peter; Geng, Xuehui; Li, Lehong



A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P.  


Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules. PMID:24332032

Puhar, Andrea; Tronchère, Hélène; Payrastre, Bernard; Nhieu, Guy Tran Van; Sansonetti, Philippe J



Definition of phenotype.  


Definition of the phenotype is crucial in designing any genetic study, especially an association study, intended to detect the disease predisposing genes. In this chapter, we review the different types of phenotypes such as discrete or continuous and discuss the issues impacting on the phenotype definition related to study design, specifically, the impact of diagnostic error (misclassification) in case-control studies and measurement error in continuous traits. We show that the power of a study depends heavily on the phenotype measured and that misclassification or measurement error can dramatically reduce the power. We also suggest some possible responses to these challenges. PMID:18358317

Wojczynski, Mary K; Tiwari, Hemant K



SEDS experiment design definition  

NASA Technical Reports Server (NTRS)

The Small Expendable-tether Deployment System (SEDS) was developed to design, build, integrate, fly, and safely deploy and release an expendable tether. A suitable concept for an on-orbit test of SEDS was developed. The following tasks were performed: (1) Define experiment objectives and requirements; (2) Define experiment concepts to reach those objectives; (3) Support NASA in experiment concept selection and definition; (4) Perform analyses and tests of SEDS hardware; (5) Refine the selected SEDS experiment concept; and (6) Support interactive SEDS system definition process. Results and conclusions are given.

Carroll, Joseph A.; Alexander, Charles M.; Oldson, John C.



Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP  

PubMed Central

Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into ?22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg2+ or performing the reaction at 4°C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4°C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.

Zhang, Haidi; Kolb, Fabrice A.; Brondani, Vincent; Billy, Eric; Filipowicz, Witold



Kinetics of PCr to ATP and ?-ATP to ?-ADP phosphoryl conversion are modified in working rat skeletal muscle after training  

Microsoft Academic Search

Kinetics of phosphoryl transfers from PCr to ?-ATP and from ?-ATP to ?-ADP were measured by magnetization transfer in an in\\u000a vivo31P NMR experiment in working rat skeletal hind leg muscles. Two groups were examined. One group was submitted to a 6-week training\\u000a program of treadmill running. The other group was composed of sedentary animals. Metabolic oxidative capacity and mechanical

Xavier Ravalec; Nathalie Le Tallec; François Carré; Jacques D. de Certaines; Elisabeth Le Rumeur



Aminoacyl-tRNA Synthetases: Affinity Labeling of the ATP Binding Site by 2',3'-ribose Oxidized ATP  

Microsoft Academic Search

Homogeneous Escherichia coli methionyl-, isoleucyl-, tryptophanyl-, and phenylalanyl-tRNA synthetases and Bacillus stearothermophilus methionyl- and tyrosyl-tRNA synthetases are irreversibly inactivated by reaction of their active ATP sites with the 2',3'-dialdehyde derivative of ATP obtained by periodate oxidation. In each case, the amount of 14C-labeled dialdehyde derivative incorporated per molecule of inactivated enzyme appears consistent with the expected active stoichiometry of the

Guy Fayat; Michel Fromant; Sylvain Blanquet



Co-activation of P2Y2 Receptor and TRPV Channel by ATP: Implications for ATP Induced Pain  

Microsoft Academic Search

Summary 1.Extracellular ATP is recognized as a peripheral modulator of pain. Activation of ionotropic P2X receptors in sensory neurons has been implicated in induction of pain, whereas metabotropic P2Y receptors in potentiation of pain induced by chemical or physical stimuli via capsaicin sensitive TRPV1 channel. Here we report that P2Y2 receptor activation by ATP can activate the TRPV1 channel in

Srihasam Lakshmi; Preeti G. Joshi



Photovoltaic Applications Definition and Photovoltaic System Definition Study in the Agricultural Sector. Volume III. Appendixes.  

National Technical Information Service (NTIS)

The appendices presented in this volume include: (1) description of energy consumption for representative farm operations; (2) design and simulation of ventilation for livestock shelters; (3) irrigation systems and calculations; (4) detailed methodology f...

R. W. Mengel T. P. Nadolski D. C. Sparks S. K. Young A. Yingst



positive definite Toeplitz matrix  

Microsoft Academic Search

Recent progress in signal processing and estimation has gener- ated considerable interest in the problem of computing the smallest eigenvalue of symmetric positive definite Toeplitz matrices. Several algorithms have been proposed in the literature. Many of them com- pute the smallest eigenvalue in an iterative fashion, relying on the Levinson-Durbin solution of sequences of Yule-Walker systems. Exploiting the properties of

Nicola Mastronardi; Marc Van Barel; Raf Vandebril



ERIC Educational Resources Information Center




VSCE technology definition study  

NASA Technical Reports Server (NTRS)

Refined design definition of the variable stream control engine (VSCE) concept for advanced supersonic transports is presented. Operating and performance features of the VSCE are discussed, including the engine components, thrust specific fuel consumption, weight, noise, and emission system. A preliminary engine design is presented.

Howlett, R. A.; Hunt, R. B.



The definition of abstinence.  


A teen's definition of sexual activity most often does not include oral or anal sex. Abstinence only programs vary widely as to how they define sexual behavior and may be contributing to misinformation about STD transmission. Unknown is the extent to which declining teen pregnancy rates are due to non-coital activities replacing vaginal intercourse. PMID:15749587

Nicoletti, Angela



A Definition of Planet  

Microsoft Academic Search

It had proposed some definitions about what a planet is. It seems clear that the planet's mass superior limit should be lower than the threshold for deuterium thermonuclear fusion. However the inferior limit is more elusive. It had proposed either Pluto's mass or the minimum mass to produce a spherical form. The Working Group on Extrasolar Planets (WGESP) of the

H. J. Durand-Manterola



Pannexin 1 Contributes to ATP Release in Airway Epithelia  

PubMed Central

ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air–liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (±64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (±0.4) ?M, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n ? 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 ?M) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 ?M), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.

Ransford, George A.; Fregien, Nevis; Qiu, Feng; Dahl, Gerhard; Conner, Gregory E.; Salathe, Matthias



Regulation of yeast acetohydroxyacid synthase by valine and ATP.  

PubMed Central

The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity. It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes. One type of subunit contains the catalytic machinery, whereas the other has a regulatory function. Previously, we have shown [Pang and Duggleby (1999) Biochemistry 38, 5222--5231] that yeast AHAS can be reconstituted from its separately purified subunits. The reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS. High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits. It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5'-[beta,gamma-imido]triphosphate. The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model.

Pang, S S; Duggleby, R G



An enhanced molecular dynamics study of HPPK-ATP conformation space exploration and ATP binding to HPPK.  


HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyzes the transfer of pyrophosphate from ATP to HP (6-hydroxymethyl-7,8-dihydropterin). This first reaction in the folate biosynthetic pathway is an important target for potential antimicrobial agents. In this work, the mechanism by which HPPK traps and binds ATP is studied by molecular dynamics (MD)-based methods. Based on the ternary crystal structure of HPPK with an ATP mimic and HP, a complex of ATPMg(2) and HPPK is simulated and found to undergo small conformational changes with conventional MD, as does also conventional MD when started from the apo crystal structure. The introduction of restraints in the MD that serve to move HPPK-ATP from its ternary complex (closed) to apo-like (open) forms shows that throughout the restraint path ATP remains bound to HPPK. That ATP remains bound suggests that there is an ensemble of conformations with ATP bound to HPPK that span the apo to more ligand-bound-like conformations, consistent with the pre-existing equilibrium hypothesis of ligand binding, whereby a ligand can select from and bind to a broad range of protein conformations. In the apo-like conformations, ATPMg(2) remains bound to HPPK through a number of mainly salt-bridge-like interactions between several negatively charged residues and the two magnesium cations. The introduction of a reweight method that enhances the sampling of MD by targeting explicit terms in the force field helps define the interactions that bind ATP to HPPK. Using the reweight method, conformational and center of mass motions of ATP, driven by the breaking and making of hydrogen bonds and salt bridges, are identified that lead to ATP separating from HPPK. An elastic normal mode (ENM) approach to opening the ternary complex and closing the apo crystal structures was carried out. The ENM analysis of the apo structure analysis shows one mode that does have a closing motion of HPPK loops, but the direction does not correlate strongly with the loop motions that are required for forming the ternary complex. PMID:19191740

Su, Li; Cukier, Robert I



ATP P2X3 receptors and neuronal sensitization  

PubMed Central

Increasing evidence indicates the importance of extracellular adenosine triphosphate (ATP) in the modulation of neuronal function. In particular, fine control of ATP release and the selective and discrete ATP receptor operation are crucial elements of the crosstalk between neuronal and non-neuronal cells in the peripheral and central nervous systems. In peripheral neurons, ATP signaling gives an important contribution to neuronal sensitization, especially that involved in neuropathic pain. Among other subtypes, P2X3 receptors expressed on sensory neurons are sensitive even to nanomolar concentrations of extracellular ATP, and therefore are important transducers of pain stimuli. P2X3 receptor function is highly sensitive to soluble factors like neuropeptides and neurotrophins, and is controlled by transduction mechanisms, protein-protein interactions and discrete membrane compartmentalization. More recent findings have demonstrated that P2X3 receptors interact with the synaptic scaffold protein calcium/calmodulin-dependent serine protein kinase (CASK) in a state dependent fashion, indicating that CASK plays a crucial role in the modulation of P2X3 receptor stability and efficiency. Activation of P2X3 receptors within CASK/P2X3 complex has important consequences for neuronal plasticity and possibly for the release of neuromodulators and neurotransmitters. Better understanding of the interactome machinery of P2X3 receptors and their integration with other receptors and channels on neuronal surface membranes, is proposed to be essential to unveil the process of neuronal sensitization and related, abnormal pain signaling.

Fabbretti, Elsa



Mechanisms of ATP Release, the Enabling Step in Purinergic Dynamics  

PubMed Central

The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A3ARs in the ciliary epithelium activate Cl? channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A1ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X7 release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.

Li, Ang; Banerjee, Juni; Leung, Chi Ting; Peterson-Yantorno, Kim; Stamer, W. Daniel; Civan, Mortimer M.



ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry  

PubMed Central

Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F1Fo-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F1Fo-ATP synthases. Under selected buffer conditions the mass of the intact F1Fo-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases.

Hoffmann, Jan; Sokolova, Lucie; Preiss, Laura; Hicks, David B.; Krulwich, Terry A.; Morgner, Nina; Wittig, Ilka; Schagger, Hermann; Meier, Thomas; Brutschy, Bernd



Rotation and structure of FoF1-ATP synthase.  


F(o)F(1)-ATP synthase is one of the most ubiquitous enzymes; it is found widely in the biological world, including the plasma membrane of bacteria, inner membrane of mitochondria and thylakoid membrane of chloroplasts. However, this enzyme has a unique mechanism of action: it is composed of two mechanical rotary motors, each driven by ATP hydrolysis or proton flux down the membrane potential of protons. The two molecular motors interconvert the chemical energy of ATP hydrolysis and proton electrochemical potential via the mechanical rotation of the rotary shaft. This unique energy transmission mechanism is not found in other biological systems. Although there are other similar man-made systems like hydroelectric generators, F(o)F(1)-ATP synthase operates on the nanometre scale and works with extremely high efficiency. Therefore, this enzyme has attracted significant attention in a wide variety of fields from bioenergetics and biophysics to chemistry, physics and nanoscience. This review summarizes the latest findings about the two motors of F(o)F(1)-ATP synthase as well as a brief historical background. PMID:21524994

Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki



Capture and quality control mechanisms for ATP binding  

PubMed Central

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly-casting mechanism that acts up on the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates.

Li, Li; Martinis, Susan A.



On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP  

PubMed Central

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.

Birchmeier, W; Singer, SJ



Heat shock protein 70 (Hsp70) inhibits oxidative phosphorylation and compensates ATP balance through enhanced glycolytic activity  

PubMed Central

To address possible effects of heat shock protein 70 (Hsp70) on energy metabolism, we established a cell line expressing different levels of Hsp70 and evaluated changes in glucose and lactate metabolites, as well as ATP levels accordingly. In addition, activities of enzymes involved in glycolysis [phosphofructokinase (PFK) and lactate dehydrogenase (LDH)], Krebs cycle [citric synthase (CS)], and oxidative phosphorylation {NADH dehydrogenase [complex I (CI)] and ubiquinol:cytochrome-c reductase [complex III (CIII)]} were analyzed. The results show that both glucose consumption and lactate excretion were elevated significantly in cells expressing increased levels of Hsp70. Simultaneously, the activities of glycolytic enzymes PFK and LDH were increased markedly in cells overexpressing Hsp70. Activities of enzymes CI and CIII, both involved in oxidative phosphorylation, decreased upon increased expression of Hsp70. These findings were supported by nonsignificant reductions of CS activities in cells that overexpressed Hsp70, whereas intracellular ATP levels remained constant over a wide range of Hsp70 expression. In conclusion, overexpression of Hsp70 in HeLa cells results in downregulation of oxidative phosphorylation, in particular, multiprotein CIII, the main source of reactive oxygen species. In exchange, upregulation of the glycolytic pathway compensates for the homeostasis of cellular ATP supply.

Wang, Liangli; Schumann, Uwe; Prokopchuk, Olga; Steinacker, Jurgen M.



Structural Basis for Substrate Binding and the Catalytic Mechanism of Type III Pantothenate Kinase  

SciTech Connect

Pantothenate kinase (PanK) catalyzes the first step of the universal five-step coenzyme A (CoA) biosynthetic pathway. The recently characterized type III PanK (PanK-III, encoded by the coaX gene) is distinct in sequence, structure and enzymatic properties from both the long-known bacterial type I PanK (PanK-I, exemplified by the Escherichia coli CoaA protein) and the predominantly eukaryotic type II PanK (PanK-II). PanK-III enzymes have an unusually high K{sub m} for ATP, are resistant to feedback inhibition by CoA, and are unable to utilize the N-alkylpantothenamide family of pantothenate analogues as alternative substrates, thus making type III PanK ineffective in generating CoA analogues as antimetabolites in vivo. Previously, we reported the crystal structure of the PanK-III from Thermotoga maritima and identified it as a member of the 'acetate and sugar kinase/heat shock protein 70/actin' (ASKHA) superfamily. Here we report the crystal structures of the same PanK-III in complex with one of its substrates (pantothenate), its product (phosphopantothenate) as well as a ternary complex structure of PanK-III with pantothenate and ADP. These results are combined with isothermal titration calorimetry experiments to present a detailed structural and thermodynamic characterization of the interactions between PanK-III and its substrates ATP and pantothenate. Comparison of substrate binding and catalytic sites of PanK-III with that of eukaryotic PanK-II revealed drastic differences in the binding modes for both ATP and pantothenate substrates, and suggests that these differences may be exploited in the development of new inhibitors specifically targeting PanK-III.

Yang, Kun; Strauss, Erick; Huerta, Carlos; Zhang, Hong (Stellenbosch); (UTSMC)



A Consistent Definition for Quantiles.  

ERIC Educational Resources Information Center

Considers definitions of quantiles. Describes median and quartiles. Compares the usefulness of 3 different definitions of quartile using a computer program to simulate 500 quantiles on a sample of a fixed size. Five references are listed. (YP)

Fleet, Tony



ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.  


Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

Randak, Christoph O; Dong, Qian; Ver Heul, Amanda R; Elcock, Adrian H; Welsh, Michael J



Thermal activation and ATP dependence of the cytoskeleton remodeling dynamics  

NASA Astrophysics Data System (ADS)

The cytoskeleton (CSK) is a nonequilibrium polymer network that uses hydrolyzable sources of free energy such as adenosine triphosphate (ATP) to remodel its internal structure. As in inert nonequilibrium soft materials, CSK remodeling has been associated with structural rearrangements driven by energy-activated processes. We carry out particle tracking and traction microscopy measurements of alveolar epithelial cells at various temperatures and ATP concentrations. We provide the first experimental evidence that the remodeling dynamics of the CSK is driven by structural rearrangements over free-energy barriers induced by thermally activated forces mediated by ATP. The measured activation energy of these forces is ˜40kBTr ( kB being the Boltzmann constant and Tr being the room temperature). Our experiments provide clues to understand the analogy between the dynamics of the living CSK and that of inert nonequilibrium soft materials.

Sunyer, R.; Ritort, F.; Farré, R.; Navajas, D.



Modulation of proton pumping efficiency in bacterial ATP synthases.  


The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled. PMID:16765908

Turina, Paola; Rebecchi, Alberto; D'Alessandro, Manuela; Anefors, Sofie; Melandri, B Andrea



Measurement of ATP in single oocytes: impact of maturation and cumulus cells on levels and consumption.  


Mitochondria provide the primary source of ATP in the oocyte and early embryo and mitochondrial dysfunction and deficit of mitochondria-derived ATP has been linked to suboptimal developmental competence. We have undertaken a study of ATP in the maturing mouse oocyte using a novel recombinant FRET based probe, AT1.03. We show that AT1.03 can be successfully used to monitor cytosolic ATP levels in single live oocytes over extended time periods. We find that ATP levels undergo dynamic changes associated with specific maturational events and that oocytes display altered rates of ATP consumption at different stages of maturation. Cumulus enclosed oocytes have a higher ATP level during maturation than denuded oocytes and this can be abolished by inhibition of gap junctional communication between the oocyte and cumulus cells. Our work uses a new approach to shed light on regulation of ATP levels and ATP consumption during oocyte maturation. PMID:24002908

Dalton, Caroline M; Szabadkai, Gyorgy; Carroll, John



NPY mediates ATP-induced neuroproliferation in adult mouse olfactory epithelium  

PubMed Central

In the CNS, ATP is released upon injury and promotes neuroproliferation via purinergic receptors. In the olfactory epithelium, ATP promotes the synthesis and release of neurotrophic factor NPY in neonates and induces neuroproliferation in neonatal and adult mice. We tested the hypothesis that NPY is involved in ATP-induced neuroproliferation in adult mice olfactory epithelium. Intranasal instillation of ATP significantly increased protein levels and number of NPY+ cells. Pre-intranasal instillation of purinergic receptor antagonist PPADS significantly reduced ATP-induced upregulation of NPY. Intranasal instillation of NPY-Y1 receptor antagonist BIBP3226 following ATP instillation significantly inhibited the ATP-induced increase in BrdU incorporation, suggesting that NPY is released after ATP instillation and activates Y1 receptors to promote neuroproliferation. These data indicate that ATP initiates neuroproliferation via NPY upregulation, NPY release, and Y1 receptor activation, and suggests that the olfactory epithelium is good model to study neuroregenerative mechanisms in the CNS.

Jia, Cuihong; Hegg, Colleen Cosgrove



NASA assurance terms and definitions  

NASA Technical Reports Server (NTRS)

This publication provides a compendium of commonly used safety, reliability, maintainability, and quality assurance (SRM&QA) definitions to ensure standardized assurance communications among NASA Field Installations, Headquarters, and contractors. This list of standard assurance terms and definitions shall be utilized by all NASA organizations and contractors. Program/project tailoring of these definitions may be permitted for specific program applications.



State Definitions of Emotional Disturbance  

ERIC Educational Resources Information Center

This article examines definitions state education agencies use to describe the federal education disability called "emotional disturbance." State definitions were collected so that various aspects of them could be analyzed and compared with results of similar studies completed in the 1970s and 1980s. Among results are that state definitions have…

Wery, Jessica J.; Cullinan, Douglas



Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli  

PubMed Central

Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases.

Jaramillo, Michael L; Abanto, Michel; Quispe, Ruth L; Calderon, Julio; del Valle, Luis J; Talledo, Miguel; Ramirez, Pablo



Synthesis and in vitro characterization of a novel PAA-ATP conjugate.  


The objective of this study was to improve the multifunctional properties of poly(acrylic acid) (PAA) by covalent attachment of 4-aminothiophenol (ATP) to its backbone. The permeation enhancing effect of PAA-ATP together with glutathione was evaluated in Ussing-type chambers using fluorescein isothiocyanate dextran as model compound. The mucoadhesive properties were evaluated in vitro on freshly excised porcine intestinal mucosa through the rotating cylinder method. The resulting conjugates PAA-ATP1 and PAA-ATP2 displayed 168 ± 35 and 426 ± 55 ?mol immobilized free thiol groups per gram polymer, respectively. In addition, 279 ± 28 and 139 ± 22 ?mol disulfide bonds per gram polymer, respectively, were identified on PAA-ATP1 and PAA-ATP2. Within disintegration studies in aqueous buffer solution, the modified polymers showed improved cohesive properties. Because of the immobilization of ATP, the swelling of PAA-ATP1 and PAA-ATP2 improved 12.0- and 17.8-fold, respectively. The adhesion times of the conjugates PAA-ATP1 and PAA-ATP2 were more than 20- and 30-fold increased in comparison to unmodified PAA. Furthermore, conjugates PAA-ATP1 and PAA-ATP2 exhibited a 1.86- and 2.07-fold higher permeation enhancing effect, respectively, over unmodified PAA. According to these results, PAA-ATP conjugates represent a very promising novel type of thiomer for the development of various mucoadhesive drug delivery systems. PMID:20923387

Hoyer, Herbert; Hombach, Juliane; Perera, Glen; Thaurer, Michael; Bernkop-Schnürch, Andreas



Processing mechanics of alternate twist ply (ATP) yarn technology  

NASA Astrophysics Data System (ADS)

Ply yarns are important in many textile manufacturing processes and various applications. The primary process used for producing ply yarns is cabling. The speed of cabling is limited to about 35m/min. With the world's increasing demands of ply yarn supply, cabling is incompatible with today's demand activated manufacturing strategies. The Alternate Twist Ply (ATP) yarn technology is a relatively new process for producing ply yarns with improved productivity and flexibility. This technology involves self plying of twisted singles yarn to produce ply yarn. The ATP process can run more than ten times faster than cabling. To implement the ATP process to produce ply yarns there are major quality issues; uniform Twist Profile and yarn Twist Efficiency. The goal of this thesis is to improve these issues through process modeling based on understanding the physics and processing mechanics of the ATP yarn system. In our study we determine the main parameters that control the yarn twist profile. Process modeling of the yarn twist across different process zones was done. A computational model was designed to predict the process parameters required to achieve a square wave twist profile. Twist efficiency, a measure of yarn torsional stability and bulk, is determined by the ratio of ply yarn twist to singles yarn twist. Response Surface Methodology was used to develop the processing window that can reproduce ATP yarns with high twist efficiency. Equilibrium conditions of tensions and torques acting on the yarns at the self ply point were analyzed and determined the pathway for achieving higher twist efficiency. Mechanistic modeling relating equilibrium conditions to the twist efficiency was developed. A static tester was designed to zoom into the self ply zone of the ATP yarn. A computer controlled, prototypic ATP machine was constructed and confirmed the mechanistic model results. Optimum parameters achieving maximum twist efficiency were determined in this study. The successful results of this work have led to the filing of a US patent disclosing the method for producing ATP yarns with high yarn twist efficiency using a high convergence angle at the self ply point together with applying ply torque.

Elkhamy, Donia Said


Elucidation of the ATP7B N-Domain Mg2+-ATP Coordination Site and Its Allosteric Regulation  

PubMed Central

The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain) of the Mg2+-ATP coordination site and answer to the controversial role of the Mg2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process.

Hercend, Claude; Bauvais, Cyril; Bollot, Guillaume; Delacotte, Nicolas; Chappuis, Philippe; Woimant, France; Launay, Jean-Marie; Manivet, Philippe




USGS Publications Warehouse

Talc is a naturally occurring single-phase mineral having the approximate chemical formula Mg//3Si//4O//1//0(OH)//2 and a specific type of crystal structure. Talc commonly forms by hydrothermal alteration of rocks rich in magnesium and iron (ultramafic rocks) and by low-grade thermal metamorphism of siliceous dolomites. The fact that talc often occurs in association with other minerals, or that its crystalline form may be platy, fibrous, acicular, or massive, does not modify its definition. The paper discusses variations in talc chemical composition and its crystal structure and describes its characteristic physical properties.

Ross, Malcolm



Quantum and classical dynamics simulations of ATP hydrolysis in solution  

PubMed Central

ATP hydrolysis is a key reaction in living cells that drives many cellular processes. The reaction, which involves gamma phosphate cleavage from ATP, converting it to ADP, has been suggested to occur via an associative or dissociative mechanism dependent upon the surrounding environment. Prior quantum chemical studies suffered from short simulation timescales failing to capture free energy contributions due to relaxation of the surrounding aqueous environment. We have developed a highly parallelized QM/MM implementation in the NAMD and OpenAtom simulation packages, using the dual grid, dual length scale method for combined plane-wave and Eular exponential spline-based QM/MM simulations. This approach, using message-driven parallel quantum and classical dynamics, permits sufficient timescale simulations for quantum chemical events such as ATP hydrolysis, and is found to accurately and reliably include the free energy contributions of solvent relaxation to hydrolysis. In this paper we describe the application of the dual grid, dual length plane-wave-based QM/MM method to study both the associative and dissociative mechanisms of ATP hydrolysis, accounting for the free energy contribution from solvent relaxation, as well as for the key role of Mg2+ in the reaction.

Harrison, Christopher B.; Schulten, Klaus



ATP synthase inhibition of Mycobacterium avium is not bactericidal.  


The efficacy of ATP synthase inhibitor TMC207 was assessed in early and late Mycobacterium avium infections in mice. In contrast to what was earlier observed for M. tuberculosis, a bacteriostatic effect was obtained. In vitro, the minimal bactericidal concentration (MBC)/MIC ratio was very high. The MBC was more relevant for assessment of pharmacokinetic/pharmacodynamic relationships than the MIC. PMID:19738016

Lounis, Nacer; Gevers, Tom; Van den Berg, Joke; Vranckx, Luc; Andries, Koen



ATP binding turns plant cryptochrome into an efficient natural photoswitch.  


Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction. PMID:24898692

Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D; Ritz, Thorsten; Brettel, Klaus



Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase  

PubMed Central

Summary When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca2+ regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After ? -actin, the cytosolic brain isoform of creatine kinase was the second-most abundant bundle protein; at ~0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca2+-ATPase. Consistent with this critical role in hair-bundle function, the creatine kinase circuit is essential for high-sensitivity hearing, as demonstrated by hearing loss in creatine kinase knockout mice.

Shin, Jung-Bum; Streijger, Femke; Beynon, Andy; Peters, Theo; Gadzalla, Laura; McMillen, Debra; Bystrom, Cory; Van der Zee, Catharina E.E.M; Wallimann, Theo; Gillespie, Peter G.



ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch  

PubMed Central

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH· radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·?, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396?. Its negative charge could trigger conformational changes necessary for signal transduction.

Muller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Veronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus



Electromagnetic field generation by ATP-induced reverse electron transfer  

Microsoft Academic Search

This paper describes a mechanism to explain low-level light emission in biology. A biological analog of the electrical circuitry, modeled on the parallel plate capacitor, traversed by a helical structure, required to generate electromagnetic radiation in the optical spectral range, is described. The charge carrier required for the emissions is determined to be an accelerating electron driven by an ATP-induced

Richard H. Steele




NSDL National Science Digital Library

ATPase, proton-transporting, lysosomal V0 subunit D1 (ATP6V0D1, also known as A0403) is responsible for the acidification of endosomes, lysosomes, and other intracellular organelles in eukaryotic cells, thus providing most of the energy required for ...



Kinetics of signaling-DNA-aptamer-ATP binding  

Microsoft Academic Search

DNA aptamers are molecular biosensors consisting of single functionalized DNA molecules, which can bind to specific targets or complementary DNA sequences. The binding kinetics of DNA aptamers is studied by fluorescence quenching at 23°C . A kinetic model for the binding reaction of DNA aptamer, antisense DNA, and ATP target is developed to describe experimental observations. The approach leads to

Issei Nakamura; An-Chang Shi; Razvan Nutiu; Jasmine M. Y. Yu; Yingfu Li



Cyclodextrin-based microcapsules as bioreactors for ATP biosynthesis.  


A biomimetic energy converter was fabricated via the assembly of CF0F1-ATPase on lipid-coated hollow nanocapsules composed of ?-cyclodextrins/chitosan-graft-poly(ethylene glycol) methacrylate. Upon entrapped GOD into these capsules, the addition of glucose could trigger proton-motive force and then drive the rotation of ATPase to synthesize ATP. PMID:23962233

Li, Jian-Hu; Wang, Yi-Fu; Ha, Wei; Liu, Yan; Ding, Li-Sheng; Li, Bang-Jing; Zhang, Sheng



Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase  

Microsoft Academic Search

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation,

Jung-Bum Shin; Femke Streijger; Andy Beynon; Theo Peters; Laura Gadzala; Debra McMillen; Cory Bystrom; Catharina E. E. M. Van der Zee; Theo Wallimann; Peter G. Gillespie



Developmental aspects of amperometric ATP biosensors based on entrapped enzymes.  


A novel concept for a dual-enzyme-based microbiosensor for the detection of adenosine-5'-triphosphate (ATP) was developed. The employed enzymes pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and hexokinase were entrapped, using pH-shift-induced precipitation of electrodeposition paint (EDP) at platinum microelectrodes (diameter of 25 microm). PQQ-GDH is known showing a superior activity for glucose conversion at the relevant conditions (low oxygen concentration) for ATP detection in targeted biomedical studies. For immobilizing the two enzymes PQQ-GDH and hexokinase, the deposition conditions of EDP Resydrol AY498w/35WA were adapted to ensure high immobilization rates. Prior to ATP sensing, the conversion of glucose, which is the co-substrate for both enzymatic reactions, was optimized. Optimization was targeted towards ATP measurements in biomedical environments by optimizing the PQQ-GDH sensor for glucose. Therefore, different mediators were tested regarding their electron transfer rate and their compatibility with the enzyme: free-diffusing N-methylphenazonium methyl sulfate (PMS) and ferrocenemethanol, and an immobilized chromium hexacyanoferrate layer at platinum electrode. Free-diffusing ferrocenemethanol reveals high sensitivity towards glucose of 1.5 +/- 0.4 nA/mM. In a next step, hexokinase was co-entrapped in the polymer film resulting in a sensitivity of up to 290 pA/microM. PMID:19779927

Weber, Cornelia; Gauda, Estelle; Mizaikoff, Boris; Kranz, Christine



P2Y Receptor Modulation of ATP Release in the Urothelium  

PubMed Central

The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P < 0.0011, ADP EC50 1.8?µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62?µM). Selective P2 receptor agonists, ??-methylene-ATP, ATP-?-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin (100?µM, P = 0.002). RT4 urothelial cells break down nucleotides (100?µM) including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling.

Mansfield, Kylie J.; Hughes, Jessica R.



KATP-channels in beta-cells in tissue slices are directly modulated by millimolar ATP.  


In pancreatic beta-cells, inhibition of K(ATP)-channels plays a pivotal role in signal transduction of glucose-induced insulin release. However, the extreme sensitivity of K(ATP)-channels to its ligand ATP as found in inside-out patches is not directly compatible with modulation of these channels at physiological [ATP](i). We studied K(ATP)-channel sensitivity to ATP in beta-cells in dispersed culture and in fresh pancreatic tissue slices. Physiological [ATP](i) blocks more than 99% of K(ATP)-channels in cultured beta-cells, while only 90% in beta-cells in slices, indicating reduced sensitivity to ATP in the fresh slices. Applying cytosolic factors like ADP, phosphatidylinositol-4,5-bisphosphate (PIP(2)) or oleoyl-CoA did not restore the K(ATP)-channel sensitivity in cultured beta-cells. Our data suggest that interaction between SUR1 and Kir6.2 subunit of the K(ATP)-channel could be a factor in sensitivity modulation. Tissue slices are the first beta-cell preparation to study direct K(ATP)-channel modulation by physiological [ATP](i). PMID:15664451

Speier, S; Yang, S-B; Sroka, K; Rose, T; Rupnik, M



Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport  

NASA Technical Reports Server (NTRS)

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.



Type III or allosteric kinase inhibitors for the treatment of non-small cell lung cancer.  


Introduction: In recent times, there has been much interest in the development of pharmacological kinase inhibitors that treat NSCLC. Furthermore, treatment options have been guided by the development of a wide panel of synthetic small molecule kinase inhibitors. Most of the molecules developed belong to the type I class of inhibitors that target the ATP-binding site in its active conformation. The high sequence similarity in the ATP-binding site among members of the kinase families often results in low selectivity and additional toxicities. Also, second mutations in the ATP-binding site, such as threonine to methionine at position 790, have been described as a mechanism of resistance to ATP-competitive kinase inhibitors. For these reasons, alternative drug development approaches targeting sites other than the ATP cleft are being pursued. The class III or allosteric inhibitors, which bind outside the ATP-binding site, have been shown to negatively modulate kinase activity. Areas covered: In this review, the authors discuss the most well-characterised allosteric inhibitors that have reached clinical development in NSCLC. Expert opinion: Great progress has made in developing inhibitors with entirely new modes of action. That being said, it is important to highlight that despite their apparent simplicity, biochemical assays will remain at the core of drug discovery activities to better explore these new opportunities. PMID:24673358

Fasano, Morena; Della Corte, Carminia Maria; Califano, Raffaele; Capuano, Annalisa; Troiani, Teresa; Martinelli, Erika; Ciardiello, Fortunato; Morgillo, Floriana



77 FR 47513 - Definition of Enforcement Action  

Federal Register 2010, 2011, 2012, 2013

...Commission 25 CFR Part 502 Definition of Enforcement Action AGENCY: National Indian Gaming...definitions to add a definition of ``enforcement action.'' DATES: Effective Date...made to Part 573, a definition of ``enforcement action'' needed to be added to...



Rain radar instrument definition  

NASA Astrophysics Data System (ADS)

As a result of a pre-phase a study, founded by ESA, this paper presents the definition of a spaceborne Rain Radar, candidate instrument for earth explorer precipitation mission. Based upon the description of user requirements for such a dedicated mission, a mission analysis defines the most suitable space segment. At system level, a parametric analysis compares pros and cons of instrument concepts associated with rain rate retrieval algorithms in order to select the most performing one. Several trade-off analysis at subsystem level leads then to the definition of the proposed design. In particular, as pulse compression is implemented in order to increase the radar sensitivity, the selected method to achieve a pulse response with a side-lobe level below--60 dB is presented. Antenna is another critical rain radar subsystem and several designs are com pared: direct radiating array, single or dual reflector illuminated by single or dual feed arrays. At least, feasibility of centralized amplification using TWTA is compared with criticality of Tx/Rx modules for distributed amplification. Mass and power budgets of the designed instrument are summarized as well as standard deviations and bias of simulated rain rate retrieval profiles. The feasibility of a compliant rain radar instrument is therefore demonstrated.

Vincent, Nicolas; Chenebault, J.; Suinot, Noel; Mancini, Paolo L.



LDR structural experiment definition  

NASA Technical Reports Server (NTRS)

A study was performed to develop the definition of a structural flight experiment for a large precision segmented reflector that would utilize the Space Station. The objective of the study was to use the Large Deployable Reflector (LDR) baseline configuration for focusing on experiment definition activity which would identify the Space Station accommodation requirements and interface constraints. Results of the study defined three Space Station based experiments to demonstrate the technologies needed for an LDR type structure. The basic experiment configurations are the same as the JPL baseline except that the primary mirror truss is 10 meters in diameter instead of 20. The primary objectives of the first experiment are to construct the primary mirror support truss and to determine its structural and thermal characteristics. Addition of the optical bench, thermal shield and primary mirror segments and alignment of the optical components occur on the second experiment. The structure will then be moved to the payload pointing system for pointing, optical control and scientific optical measurement for the third experiment.

Russell, Richard A.; Gates, Richard M.



Physiological characterization of ATP-citrate lyase in Aspergillus niger.  


Acetyl-CoA, an important molecule in cellular metabolism, is generated in multiple subcellular compartments and mainly used for energy production, biosynthesis of a diverse set of molecules, and protein acetylation. In eukaryotes, cytosolic acetyl-CoA is derived mainly from the conversion of citrate and CoA by ATP-citrate lyase. Here, we describe the targeted deletions of acl1 and acl2, two tandem divergently transcribed genes encoding subunits of ATP-citrate lyase in Aspergillus niger. We show that loss of acl1 or/and acl2 results in a significant decrease of acetyl-CoA and citric acid levels in these mutants, concomitant with diminished vegetative growth, decreased pigmentation, reduced asexual conidiogenesis, and delayed conidial germination. Exogenous addition of acetate repaired the defects of acl-deficient strains in growth and conidial germination but not pigmentation and conidiogenesis. We demonstrate that both Acl1 and Acl2 subunits are required to form a functional ATP-citrate lyase in A. niger. First, deletion of acl1 or/and acl2 resulted in similar defects in growth and development. Second, enzyme activity assays revealed that loss of either acl1 or acl2 gene resulted in loss of ATP-citrate lyase activity. Third, in vitro enzyme assays using bacterially expressed 6His-tagged Acl protein revealed that only the complex of Acl1 and Acl2 showed ATP-citrate lyase activity, no enzyme activities were detected with the individual protein. Fourth, EGFP-Acl1 and mCherry-Acl2 proteins were co-localized in the cytosol. Thus, acl1 and acl2 coordinately modulate the cytoplasmic acetyl-CoA levels to regulate growth, development, and citric acid synthesis in A. niger. PMID:24566752

Chen, Hong; He, Xihong; Geng, Hongran; Liu, Hao



Activation of ATP Binding for the Autophosphorylation of DosS, a Mycobacterium tuberculosis Histidine Kinase Lacking an ATP Lid Motif*  

PubMed Central

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg440 in the DHp domain and Glu537 in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg440 and Glu537 reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik



Effect of methanol and Me 2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish ( Danio rerio)  

Microsoft Academic Search

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me2SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP\\/ATP ratios were assessed following exposure to cryoprotectants for 30min at room temperature. Results obtained

T. Zampolla; E. Spikings; T. Zhang; D. M. Rawson



Phospholipid Requirements of Ca++-Stimulated, Mg++-Dependent ATP Hydrolysis in Rat Brain Synaptic Membranes.  

National Technical Information Service (NTIS)

The phospholipid requirement for calcium ion-stimulated, magnesium ion -dependent (ATP) hydrolysis(Ca (++) /Mg (++)-ATPase) and Mg(++) -stimulated ATP hydrolysis (Mg(++)-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidatio...

C. R. Gandhi D. H. Ross J. J. Valdes R. G. Thompson



Pannexin 1 is the conduit for low oxygen tension-induced ATP release from human erythrocytes  

PubMed Central

Erythrocytes release ATP in response to exposure to the physiological stimulus of lowered oxygen (O2) tension as well as pharmacological activation of the prostacyclin receptor (IPR). ATP release in response to these stimuli requires activation of adenylyl cyclase, accumulation of cAMP, and activation of protein kinase A. The mechanism by which ATP, a highly charged anion, exits the erythrocyte in response to lowered O2 tension or receptor-mediated IPR activation by iloprost is unknown. It was demonstrated previously that inhibiting pannexin 1 with carbenoxolone inhibits hypotonically induced ATP release from human erythrocytes. Here we demonstrate that three structurally dissimilar compounds known to inhibit pannexin 1 prevent ATP release in response to lowered O2 tension but not to iloprost-induced ATP release. These results suggest that pannexin 1 is the conduit for ATP release from erythrocytes in response to lowered O2 tension. However, the identity of the conduit for iloprost-induced ATP release remains unknown.

Adderley, Shaquria P.; Bowles, Elizabeth A.; Egan, Terrance M.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.



Application of Firefly Luciferase Assay for Adenosine Triphosphate (ATP) to Antimicrobial Drug Sensitivity Testing.  

National Technical Information Service (NTIS)

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 ...

G. L. Picciolo S. Tuttle C. G. Schrock J. W. Deming M. Barza



Inter-Industry Diffusion of Technology That Results from ATP Products: Preliminary Research of the Potential Impacts of ATP Funding.  

National Technical Information Service (NTIS)

One objective of the Advanced Technology Program (ATP) is to fund research and development (R&D) in industries where the social benefit of the R&D is substantial even though the return to the individual firm performing the R&D may be low. Often these bene...

J. Popkin



HtrA2 deficiency causes mitochondrial uncoupling through the F?F?-ATP synthase and consequent ATP depletion.  


Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 ?-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice. PMID:22739987

Plun-Favreau, H; Burchell, V S; Holmström, K M; Yao, Z; Deas, E; Cain, K; Fedele, V; Moisoi, N; Campanella, M; Miguel Martins, L; Wood, N W; Gourine, A V; Abramov, A Y



Regulation of Extracellular ATP in Human Erythrocytes Infected with Plasmodium falciparum  

PubMed Central

In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A “3V” mixture containing isoproterenol (?-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ?ATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ?ATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ?ATP1 to 83–87% for h-RBCs and 63–74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300–900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ?ATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration.

Alvarez, Cora Lilia; Schachter, Julieta; de Sa Pinheiro, Ana Acacia; Silva, Leandro de Souza; Verstraeten, Sandra Viviana; Persechini, Pedro Muanis; Schwarzbaum, Pablo Julio



Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1.  

PubMed Central

17 beta-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (K(i(ED/FAS))=2.84+/-0.16 microM and K(i(ED/TAM))=100+/-2 nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (K(i(TAM))=300+/-100 nM). In ghosts, ATP (1-4 mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (K(i(ATP/ED))=2.5+/-0.23 mM, K(i(ATP/GEN))=0.99+/-0.17 mM and K(i(ATP/CB))=0.76+/-0.08 mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1 mM (K(i(ED/TAM))=130+/-5 nM and K(i(ED/FAS))=2.7+/-0.9 microM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor beta (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225-229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421-423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His(337), Arg(349) and Glu(249) at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His(475), Glu(305) and Arg(346) in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export.

Afzal, Iram; Cunningham, Philip; Naftalin, Richard J



Inhibition of chemokine expression in rat inflamed paws by systemic use of the antihyperalgesic oxidized ATP  

Microsoft Academic Search

BACKGROUND: We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP) in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. RESULTS: Local, oral or intravenous administration of

Alessandro Fulgenzi; Giacomo Dell'Antonio; Chiara Foglieni; Elena Dal Cin; Paolo Ticozzi; Josè S Franzone; Maria Elena Ferrero



Mechanical effects of muscle contraction increase intravascular ATP draining quiescent and active skeletal muscle in humans  

PubMed Central

Intravascular adenosine triphosphate (ATP) evokes vasodilation and is implicated in the regulation of skeletal muscle blood flow during exercise. Mechanical stresses to erythrocytes and endothelial cells stimulate ATP release in vitro. How mechanical effects of muscle contractions contribute to increased plasma ATP during exercise is largely unexplored. We tested the hypothesis that simulated mechanical effects of muscle contractions increase [ATP]venous and ATP effluent in vivo, independent of changes in tissue metabolic demand, and further increase plasma ATP when superimposed with mild-intensity exercise. In young healthy adults, we measured forearm blood flow (FBF) (Doppler ultrasound) and plasma [ATP]v (luciferin-luciferase assay), then calculated forearm ATP effluent (FBF×[ATP]v) during rhythmic forearm compressions (RFC) via a blood pressure cuff at three graded pressures (50, 100, and 200 mmHg; Protocol 1; n = 10) and during RFC at 100 mmHg, 5% maximal voluntary contraction rhythmic handgrip exercise (RHG), and combined RFC + RHG (Protocol 2; n = 10). [ATP]v increased from rest with each cuff pressure (range 144–161 vs. 64 ± 13 nmol/l), and ATP effluent was graded with pressure. In Protocol 2, [ATP]v increased in each condition compared with rest (RFC: 123 ± 33; RHG: 51 ± 9; RFC + RHG: 96 ± 23 vs. Mean Rest: 42 ± 4 nmol/l; P < 0.05), and ATP effluent was greatest with RFC + RHG (RFC: 5.3 ± 1.4; RHG: 5.3 ± 1.1; RFC + RHG: 11.6 ± 2.7 vs. Mean Rest: 1.2 ± 0.1 nmol/min; P < 0.05). We conclude that the mechanical effects of muscle contraction can 1) independently elevate intravascular ATP draining quiescent skeletal muscle without changes in local metabolism and 2) further augment intravascular ATP during mild exercise associated with increases in metabolism and local deoxygenation; therefore, it is likely one stimulus for increasing intravascular ATP during exercise in humans.

Crecelius, Anne R.; Kirby, Brett S.; Richards, Jennifer C.



Organisation and expression of mitochondrial atp9 genes from CMS and fertile carrots  

Microsoft Academic Search

The F0-F1 ATPase subunit 9 gene of carrot mitochondria has been isolated from both petaloid male-sterile (atp9-1) and normal fertile cytoplasms (atp9-3). The position occupied in atp9-3 by the TAA stop codon is, in the case of atp9-1 replaced by the CAA triplet coding for glutamine, which makes this latter ORF 13 amino acid residues longer than that of\\u000a the

M. Szklarczyk; M. Oczkowski; H. Augustyniak; T. Börner; B. Linke; B. Michalik



ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway  

Microsoft Academic Search

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive ?360 kDa protein\\/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active

Antti Nykänen; Benjamin Haley; Phillip D. Zamore



Involvement of anion channels in mediating elicitor-induced ATP efflux in Salvia miltiorrhiza hairy roots  

Microsoft Academic Search

This study examines the roles of anion channels and ATP binding cassette (ABC) protein transporters in mediating elicitor-induced ATP release in Salvia miltiorrhiza hairy root cultures. The elicitor-induced ATP release was effectively blocked by two putative membrane anion channel blockers, niflumic acid and Zn2+, but not by a specific Cl? channel blocker, phenylanthranilic acid. The elicitor-induced ATP release was also

Shu-Jing Wu; Ka-Chai Siu; Jian-Yong Wu



ATP pools and transients in the blue-green alga, Anabaena cylindrica  

Microsoft Academic Search

Anabaena cylindrica grown in steady state continuous culture has an extractable ATP pool, measured on the basis of the luciferin-luciferase assay of 165±35 nmoles ATP mg chla-1. This pool is maintained by a dynamic balance between the rate of ATP synthesis and the rate of ATP utilization. Phosphorylating mechanisms which can maintain the pool in the short term are total

P. J. Bottomley; W. D. P. Stewart



Continuous monitoring of ATP levels in living insulin secreting cells expressing cytosolic firefly luciferase  

Microsoft Academic Search

The second messenger role of ATP in insulin secretion was investigated in living INS-1 insulinoma cells. ATP-dependent luminescence was monitored in cells expressing high levels of firefly luciferase under the control of the tetracycline-dependent transactivator. The calibration of luminescence in permeabilized cells yielded similar ATP levels as those obtained in cell extracts with a conventional ATP assay. Stimulation of insulin

Pierre Maechler; Haiyan Wang; Claes B Wollheim



Biosynthesis of the class III lantipeptide catenulipeptin.  


Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing lanthionine and/or labionin structures. In this study, a novel class III lantipeptide termed catenulipeptin was discovered from Catenulispora acidiphila DSM 44928, and its biosynthesis was reconstituted in vitro. The multifunctional enzyme AciKC catalyzes both dehydration and cyclization of its peptide substrate AciA and installs two labionin structures in catenulipeptin. AciKC shows promiscuity with respect to cosubstrate and accepts all four NTPs. The C-terminal domain of AciKC is responsible for the labionin formation in catenulipeptin. The cyclase activity of AciKC requires the leader peptide of AciA substrate but does not require ATP or Zn(2+). Mutagenesis studies suggest that the labionin cyclization may proceed in a C-to-N-terminal direction. Catenulipeptin partially restores aerial hyphae growth when applied to surfactin-treated Streptomyces coelicolor. PMID:22725258

Wang, Huan; van der Donk, Wilfred A



Biosynthesis of the Class III Lantipeptide Catenulipeptin  

PubMed Central

Lantipeptides are ribosomally synthesized and posttranslationally modified peptides containing lanthionine and/or labionin structures. In this study, a novel class III lantipeptide termed catenulipeptin was discovered from Catenulispora acidiphila DSM 44928, and its biosynthesis was reconstituted in vitro. The multifunctional enzyme AciKC catalyzes both dehydration and cyclization of its peptide substrate AciA and installs two labionin structures in catenulipeptin. AciKC shows promiscuity with respect to cosubstrate and accepts all four NTPs. The C-terminal domain of AciKC is responsible for the labionin formation in catenulipeptin. The cyclase activity of AciKC requires the leader peptide of AciA substrate but does not require ATP or Zn2+. Mutagenesis studies suggest that the labionin cyclization may proceed in a C-to-N-terminal direction. Catenulipeptin partially restores aerial hyphae growth when applied to surfactin-treated Streptomyces coelicolor.



Product definition data interface  

NASA Technical Reports Server (NTRS)

The development and application of advanced Computer Aided Design/Computer Aided Manufacturing (CAD/CAM) technology in aerospace industry is discussed. New CAD/CAM capabilities provide the engineer and production worker with tools to produce better products and significantly improve productivity. This technology is expanding in all phases of engineering and manufacturing with large potential for improvements in productivity. The integration of CAD and CAM systematically to insure maximum utility throughout the U.S. Aerospace Industry, its large community of supporting suppliers, and the Department of Defense aircraft overhaul and repair facilities is outlined. The need for a framework for exchange of digital product definition data, which serves the function of the conventional engineering drawing is emphasized.

Birchfield, B.; Downey, P.



Definition Research Study  

NASA Technical Reports Server (NTRS)

Data were complied on the physical behavior and characteristics of plasma gas and/or dust in the context of how they relate to the self-contamination of manned orbiting vehicles. A definition is given of a systematic experimental program designed to yield the required empirical data on the plasma, neutral gas, and/or the particulate matter surrounding the orbiting vehicles associated with shuttle missions. Theoretical analyses were completed on the behavior of materials to be released from the orbiting or subsatellite shuttle vehicles. The results were used to define some general experimental design recommendations directly applicable to the space shuttle program requirement. An on-board laser probe technique is suggested for measuring the dynamic behavior, inventory, and physical characteristics of particulates in the vicinity of an orbiting spacecraft. Laser probing of cometary photodissociation is also assessed.

Marmo, F. F.; Pressman, J.



ATP monitoring technology for microbial growth control in potable water systems  

Microsoft Academic Search

ATP (Adenosine Triphosphate) is the primary energy transfer molecule present in all living biological cells on Earth. ATP cannot be produced or maintained by anything but a living organism, and as such, its measurement is a direct indication of biological activity. The main advantage of ATP as a biological indicator is the speed of the analysis - from collecting the

Patrick A. Whalen; Philip J. Whalen; James E. Cairns



Measuring ATP (Advanced Technology Program) Impact: 2006 Report on Economic Progress.  

National Technical Information Service (NTIS)

This is the second Report on Economic Progress for ATP, as we officially phase down our program. With the desire to preserve ATPs legacy, we have updated the text, tables, and report summaries to provide a comprehensive reporting on ATPs progress and to s...



Simultaneous Measurements of Action Potential Duration and Intracellular ATP in Isolated Ferret Hearts Exposed to Cyanide  

Microsoft Academic Search

Shortening of the cardiac action potential during ischemia and anoxia is likely to contribute to the decline in contractility that occurs under such conditions. It has been hypothesized that a decrease in the intracellular ATP concentration ((ATP),) underlies the changes hi the action potential. The recently discovered potassium channel activated at low ATP concentrations might provide the link between action

A. C. Elliott; G. L. Smith; D. G. Allen



Agonist trapped in ATP-binding sites of the P2X2 receptor  

PubMed Central

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent “tethering” reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 ? in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

Jiang, Ruotian; Lemoine, Damien; Martz, Adeline; Taly, Antoine; Gonin, Sophie; Prado de Carvalho, Lia; Specht, Alexandre; Grutter, Thomas



The effect of ATP analogs on posthydrolytic and force development steps in skinned skeletal muscle fibers.  

PubMed Central

ATP, 2-deoxy ATP (dATP), CTP, and UTP support isometric force and unloaded shortening velocity (Vu) to various extents (Regnier et al., Biophys. J. 74:3044-3058). Vu correlated with the rate of cross-bridge dissociation after the power stroke and the steady-state hydrolysis rate in solution, whereas force was modulated by NTP binding and cleavage. Here we studied the influence of posthydrolytic cross-bridge steps on force and fiber shortening by measuring isometric force and stiffness, the rate of tension decline (kPi) after Pi photogeneration from caged Pi, and the rate of tension redevelopment (ktr) after a sudden release and restretch of fibers. The slope of the force versus [Pi] relationship was the same for ATP, dATP, and CTP, but for UTP it was threefold less. ktr and kPi increased with increasing [Pi] with a similar slope for ATP, dATP, and CTP, but had an increasing magnitude of the relationship ATP < dATP < CTP. UTP reduced ktr but increased kPi. The results suggest that the rate constant for the force-generating isomerization increases with the order ATP < dATP < CTP < UTP. Simulations using a six-state model suggest that increasing the force-generating rate accounts for the faster kPi in dATP, CTP, and UTP. In contrast, ktr appears to be strongly affected by the rates of NTP binding and cleavage and the rate of the force-generating isomerization.

Regnier, M; Homsher, E



ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release  

PubMed Central

Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca2+ and redox insensitive, which makes it possible to monitor Ca2+-coupled ER ATP dynamics specifically. The approach uncovers a cell type–specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca2+ release is coupled to an increase of ATP within the ER. The Ca2+-coupled ER ATP increase is independent of the mode of Ca2+ mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca2+ depletion of the organelle. Our data highlight a novel Ca2+-controlled process that supplies the ER with additional energy upon cell stimulation.

Vishnu, Neelanjan; Jadoon Khan, Muhammad; Karsten, Felix; Groschner, Lukas N.; Waldeck-Weiermair, Markus; Rost, Rene; Hallstrom, Seth; Imamura, Hiromi; Graier, Wolfgang F.; Malli, Roland



Characterization of ATP-induced cell death in the GL261 mouse glioma.  


Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP-induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub-population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP-induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP-induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP-induced cell death and shed light on the importance of ATP-induced cell death in glioma development. PMID:20069573

Tamajusuku, Alessandra S K; Villodre, Emilly S; Paulus, Romela; Coutinho-Silva, Robson; Battasstini, Ana M O; Wink, Márcia R; Lenz, Guido



ATP Released by Astrocytes Mediates Glutamatergic Activity-Dependent Heterosynaptic Suppression  

Microsoft Academic Search

Extracellular ATP released from axons is known to assist activity-dependent signaling between neurons and Schwann cells in the peripheral nervous system. Here we report that ATP released from astrocytes as a result of neuronal activity can also modulate central synaptic transmission. In cultures of hippocampal neurons, endogenously released ATP tonically suppresses glutamatergic synapses via presynaptic P2Y receptors, an effect that

Jing-ming Zhang; Hui-kun Wang; Chang-quan Ye; Wooping Ge; Yiren Chen; Zheng-lin Jiang; Chien-ping Wu; Mu-ming Poo; Shumin Duan



Bullying by Definition: An Examination of Definitional Components of Bullying  

ERIC Educational Resources Information Center

Lack of definitional consensus remains an important unresolved issue within bullying research. This study examined the ability of definitional variables to predict overall level of victimisation (distress, power inequity, and provocation as predictors) and bullying (intention to harm, power inequity, and provocation as predictors) in 246…

Goldsmid, Susan; Howie, Pauline



Exogenous adenosine triphosphate (ATP) preserves proximal tubule microfilament structure and function in vivo in a maleic acid model of ATP depletion.  

PubMed Central

The hallmark of ischemic acute renal failure is a rapid and early decline in proximal tubule ATP. Since we have previously shown that over half of apical microfilament losses occur within the first 5 min of experimental ischemic injury, we postulated that microfilament (F-actin) structure and cellular location are dependent on cellular ATP levels. To test this hypothesis, we used maleic acid to selectively inhibit renal cortical ATP production in vivo. Maleic acid significantly decreased tissue ATP and apical F-actin in a dose-dependent manner relative to equimolar sodium chloride controls, yet higher doses of maleic acid quantitatively resulted in net actin polymerization, primarily in the cytoplasm. Functionally, maleic acid decreased glomerular filtration rate (GFR) and tubular reabsorption of sodium (TRNa) in a dose-dependent manner relative to sodium chloride controls. Administration of exogenous ATP resulted in significant increases in tissue ATP, net actin depolymerization, and relocation of F-actin from the cytoplasm back to the apical surface coinciding with increases in GFR and TRNa. Thus, ATP depletion induced by maleic acid resulted in significant cytoskeletal and functional alterations that were ameliorated by exogenous ATP. We therefore conclude that the structure and cellular location of F-actin necessary for normal functioning of proximal tubule cells in vivo is dependent on tissue ATP levels. Images

Kellerman, P S



The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis.  

PubMed Central

The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP-ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403-414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis.

Wu, Xueji; Yano, Mihiro; Washida, Hiroyo; Kido, Hiroshi



Sources of Intravascular ATP During Exercise in Humans: Critical Role for Skeletal Muscle Perfusion  

PubMed Central

Exercise hyperemia is regulated by several factors and one factor known to increase with exercise that evokes powerful vasomotor action is extracellular ATP. The origination of ATP detectable in plasma from exercising muscle of humans is, however, a matter of debate and ATP has been suggested to arise from sympathetic nerves, blood sources (e.g. erythrocytes), endothelial cells, and skeletal myocytes, among others. Therefore, we tested the hypothesis that acute augmentation of sympathetic nervous system activity (SNA) results in elevated plasma ATP draining skeletal muscle, and that SNA superimposition during exercise further increases ATP vs exercise alone. We show that increased SNA via ?40mmHg lower body negative pressure (LBNP) at rest does not increase plasma ATP (51±8 vs 58±7 nmol/L with LBNP), nor does it increase [ATP] above levels observed during rhythmic handgrip exercise (79±11 exercise alone vs 71±8 nmol/L with LBNP). Secondly, we tested the hypothesis that active perfusion of skeletal muscle is essential to observe increased plasma ATP during exercise. We identify that complete obstruction of blood flow to contracting muscle abolishes exercise-mediated increases in plasma ATP (90±19 to 49±12 nmol/L), and further, that cessation of blood flow prior to exercise completely inhibits the typical rise in ATP (3 vs 61%; obstructed vs intact perfusion). The lack of ATP change during occlusion occurred in the face of continued muscle work and elevated SNA, indicating the rise of intravascular ATP is not resultant from these extravascular sources. Our collective observations indicate that the elevation in extracellular ATP observed in blood during exercise is unlikely to originate from sympathetic nerves or the contacting muscle itself, but rather is dependent on intact skeletal muscle perfusion. We conclude that an intravascular source for ATP is essential and points toward an important role for blood sources (e.g. red blood cells) in augmenting and maintaining elevated plasma ATP during exercise.

Kirby, Brett S.; Crecelius, Anne R.; Richards, Jennifer C.; Dinenno, Frank A.



Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP.  


Recent studies have shown that heterozygous mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, cause permanent neonatal diabetes either alone (R201C, R201H) or in association with developmental delay, muscle weakness and epilepsy (V59G,V59M). Functional analysis in the absence of Mg2+, to isolate the inhibitory effects of ATP on Kir6.2, showed that both types of mutation reduce channel inhibition by ATP. However, in pancreatic beta-cells, K(ATP) channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by an auxiliary subunit, the sulphonylurea receptor SUR1. We therefore studied the MgATP sensitivity of KCNJ11 mutant K(ATP) channels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg2+ dramatically reduced the ATP sensitivity of heterozygous R201C, R201H, V59M and V59G channels. This effect was predominantly mediated via the nucleotide-binding domains of SUR1 and resulted from an enhanced stimulatory action of MgATP. Our results therefore demonstrate that KCNJ11 mutations increase the current magnitude of heterozygous K(ATP) channels in two ways: by increasing MgATP activation and by decreasing ATP inhibition. They further show that the fraction of unblocked K(ATP) current at physiological MgATP concentrations correlates with the severity of the clinical phenotype. PMID:16087682

Proks, Peter; Girard, Christophe; Ashcroft, Frances M



Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia*  

PubMed Central

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (~7 ?m) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1–10 nM. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ~250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ~1 ?M independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intra-cellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

Okada, Seiko F.; Nicholas, Robert A.; Kreda, Silvia M.; Lazarowski, Eduardo R.; Boucher, Richard C.



The National Cholesterol Education Program Adult Treatment Panel III guidelines.  


Coronary heart disease (CHD) persists as a major cause of morbidity and mortality in the United States, with more than 40% of all deaths each year directly attributed to the disease. Dyslipidemia is recognized as a major risk factor for the development and progression of CHD, with clinical trials clearly demonstrating the public health and economic benefits of favorable cholesterol modification. As a result of this evidence, the National Cholesterol Education Program (NCEP) has developed guidelines for the detection, evaluation, and treatment of high blood cholesterol in adults. The most recent of the NCEP recommendations, the Adult Treatment Panel III (ATP III) guidelines, were released in May 2001 and build on the earlier editions and reiterate the importance of low-density lipoprotein cholesterol (LDL-C) reduction to modify CHD risk. New features of the guidelines include the identification of CHD risk equivalents; lower treatment target goals; an emphasis on conditions conferring a higher risk for CHD, such as the metabolic syndrome; and a scoring system for calculating CHD risk. The ATP III emphasis on risk assessment will result in a substantial increase in the number of patients considered at risk for CHD and will expand the number eligible for lifestyle and drug intervention. PMID:14613351

Lipsy, Robert J



Structures of ATP(adenosine triphosphate)-vanadyl complexes.  


The state of existence of cytoplasmic vanadium ion is known to be important: vanadyl ion forms complexes with ATP and vanadate form inhibits (Na++K+)-ATPase. Therefore, the formation of complexes between ATP and vanadyl ion was investigated. Formations of three types of complex were observed: a blue 1:1 complex formed in acidic pH region; a blue 1:1 complex formed in neutral pH region; a green 2:1 complex formed in alkaline pH region. On the basis of the results on potentiometric titration, optical and EPR spectra and 31P- and 13C-NMR spectra, three characteristic types of coordination environment are proposed. PMID:6306604

Sakurai, H; Goda, T; Shimomura, S; Yoshimura, T



Students' interdisciplinary reasoning about "high-energy bonds" and ATP  

NASA Astrophysics Data System (ADS)

Students' sometimes contradictory ideas about ATP (adenosine triphosphate) and the nature of chemical bonds have been studied in the biology and chemistry education literatures, but these topics are rarely part of the introductory physics curriculum. We present qualitative data from an introductory physics course for undergraduate biology majors that seeks to build greater interdisciplinary coherence and therefore includes these topics. In these data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see that students' perceptions of how each scientific discipline bounds the system of interest can influence how they justify their reasoning about a topic that crosses disciplines. This has consequences for a vision of interdisciplinary education that respects disciplinary perspectives while bringing them into interaction in ways that demonstrate consistency amongst the perspectives.

Dreyfus, Benjamin W.; Geller, Benjamin D.; Sawtelle, Vashti; Svoboda, Julia; Turpen, Chandra; Redish, Edward F.



Students' interdisciplinary reasoning about "high-energy bonds" and ATP  

NSDL National Science Digital Library

Students' sometimes contradictory ideas about ATP (adenosine triphosphate) and the nature of chemical bonds have been studied in the biology and chemistry education literatures, but these topics are rarely part of the introductory physics curriculum. We present qualitative data from an introductory physics course for undergraduate biology majors that seeks to build greater interdisciplinary coherence and therefore includes these topics. In these data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see that students' perceptions of how each scientific discipline bounds the system of interest can influence how they justify their reasoning about a topic that crosses disciplines. This has consequences for a vision of interdisciplinary education that respects disciplinary perspectives while bringing them into interaction in ways that demonstrate consistency amongst the perspectives

Dreyfus, Benjamin W.; Geller, Benjamin D.; Sawtelle, Vashti; Svoboda, Julia; Turpen, Chandra; Redish, Edward F.



Diversity of operation in ATP-dependent chromatin remodelers.  


Chromatin is actively restructured by a group of proteins that belong to the family of ATP-dependent DNA translocases. These chromatin remodelers can assemble, relocate or remove nucleosomes, the fundamental building blocks of chromatin. The family of ATP-dependent chromatin remodelers has many properties in common, but there are also important differences that may account for their varying roles in the cell. Some of the important characteristics of these complexes have begun to be revealed such as their interactions with chromatin and their mechanism of operation. The different domains of chromatin remodelers are discussed in terms of their targets and functional roles in mobilizing nucleosomes. The techniques that have driven these findings are discussed and how these have helped develop the current models for how nucleosomes are remodeled. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function. PMID:21616185

Hota, Swetansu K; Bartholomew, Blaine



Structure and mechanism of ATP-binding cassette transporters  

PubMed Central

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that includes both importers and exporters. In recent years, several structures of complete ABC transporters have been determined by X-ray crystallography. These structures suggest a mechanism by which binding and hydrolysis of ATP by the cytoplasmic, nucleotide-binding domains control the conformation of the transmembrane domains and therefore which side of the membrane the translocation pathway is exposed to. A basic, conserved two-state mechanism can explain active transport of both ABC importers and ABC exporters, but various questions remain unresolved. In this article, I will review some of the crystal structures and the mechanistic insight gained from them. Future challenges for a better understanding of the mechanism of ABC transporters will be outlined.

Locher, Kaspar P.



Kinetics of signaling-DNA-aptamer-ATP binding  

NASA Astrophysics Data System (ADS)

DNA aptamers are molecular biosensors consisting of single functionalized DNA molecules, which can bind to specific targets or complementary DNA sequences. The binding kinetics of DNA aptamers is studied by fluorescence quenching at 23°C . A kinetic model for the binding reaction of DNA aptamer, antisense DNA, and ATP target is developed to describe experimental observations. The approach leads to a simple procedure to deduce relevant kinetic reactions and their rate constants. A comparison between theory and experiments indicates that the previously established bimolecular DNA-ATP binding does not provide a complete description of the experimental data. Side reactions such as trimolecular complexation are proposed. Rate constants of the model are determined by comparing the model predictions and experiments. Good agreements between the model and experiments have been obtained. Possible blocking reactions by the misfolded DNA aptamer are also discussed.

Nakamura, Issei; Shi, An-Chang; Nutiu, Razvan; Yu, Jasmine M. Y.; Li, Yingfu



Ground support equipment (GSE) for testing precision ATP payloads  

NASA Astrophysics Data System (ADS)

This paper focuses on the ground support technologies, instrumentation, and the analysis approaches employed to verify that the HABE ATP payload is correctly performing its state-of-the-art alignment, stabilization, and tracking functions. A specially constructed Laboratory Test Component (LTC) includes scoring sensors that measure the payload's LOS pointing errors by receiving and measuring the motion of the transmitted marker laser. Motion sensors with high sensitivity and broad bandwidth prove signals that are recorded simultaneously with other payload telemetry signals from the stabilized reference platform, the fast steering mirror alignment systems, and from the image track loops. The combined signals facilitate understanding and evaluation of the payload's tracking and pointing errors. With properly constructed laboratory tests and full instrumentation of the vibration and atmosphere contributed disturbances, it is possible to confirm that a sophisticated ATP payload like HABE is able to operate with residual pointing errors under a microradian.

Sebesta, Henry R.; DeMuth, Gary E.; Dillow, Michael A.; Rost, Martin C.; Laughlin, Darren R.



Toward an evolutionary definition of cheating.  


The term "cheating" is used in the evolutionary and ecological literature to describe a wide range of exploitative or deceitful traits. Although many find this a useful short hand, others have suggested that it implies cognitive intent in a misleading way, and is used inconsistently. We provide a formal justification of the use of the term "cheat" from the perspective of an individual as a maximizing agent. We provide a definition for cheating that can be applied widely, and show that cheats can be broadly classified on the basis of four distinctions: (i) whether cooperation is an option; (ii) whether deception is involved; (iii) whether members of the same or different species are cheated; and (iv) whether the cheat is facultative or obligate. Our formal definition and classification provide a framework that allow us to resolve and clarify a number of issues, regarding the detection and evolutionary consequences of cheating, as well as illuminating common principles and similarities in the underlying selection pressures. PMID:24131102

Ghoul, Melanie; Griffin, Ashleigh S; West, Stuart A



Association and Stoichiometry of K ATP Channel Subunits  

Microsoft Academic Search

ATP-sensitive potassium channels (KATP channels) are heteromultimers of sulfonylurea receptors (SUR) and inwardly rectifying potassium channel subunits (KIR6.x) with a (SUR–KIR6.x)4 stoichiometry. Association is specific for KIR6.x and affects receptor glycosylation and cophotolabeling of KIR6.x by 125I-azidoglibenclamide. Association produces digitonin stable complexes with an estimated mass of 950 kDa. These complexes can be purified by lectin chromatography or by using

John P. Clement IV; Kumud Kunjilwar; Gabriela Gonzalez; Mathias Schwanstecher; Uwe Panten; Lydia Aguilar-Bryan; Joseph Bryan



ATP as a mediator of macula densa cell signalling.  


Within each nephro-vascular unit, the tubule returns to the vicinity of its own glomerulus. At this site, there are specialised tubular cells, the macula densa cells, which sense changes in tubular fluid composition and transmit information to the glomerular arterioles resulting in alterations in glomerular filtration rate and blood flow. Work over the last few years has characterised the mechanisms that lead to the detection of changes in luminal sodium chloride and osmolality by the macula densa cells. These cells are true "sensor cells" since intracellular ion concentrations and membrane potential reflect the level of luminal sodium chloride concentration. An unresolved question has been the nature of the signalling molecule(s) released by the macula densa cells. Currently, there is evidence that macula densa cells produce nitric oxide via neuronal nitric oxide synthase (nNOS) and prostaglandin E(2) (PGE(2)) through cyclooxygenase 2 (COX 2)-microsomal prostaglandin E synthase (mPGES). However, both of these signalling molecules play a role in modulating or regulating the macula-tubuloglomerular feedback system. Direct macula densa signalling appears to involve the release of ATP across the basolateral membrane through a maxi-anion channel in response to an increase in luminal sodium chloride concentration. ATP that is released by macula densa cells may directly activate P2 receptors on adjacent mesangial cells and afferent arteriolar smooth muscle cells, or the ATP may be converted to adenosine. However, the critical step in signalling would appear to be the regulated release of ATP across the basolateral membrane of macula densa cells. PMID:19330465

Bell, P Darwin; Komlosi, Peter; Zhang, Zhi-Ren



ATP-binding cassette, subfamily G (ABCG family)  

Microsoft Academic Search

This review summarizes the characteristics of the ATP-binding cassette, subfamily G (ABCG family), which has five members:\\u000a ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8. The members consist of a single ABC cassette in the amino terminal followed by six\\u000a putative transmembrane domains, and to become functionally active, they form homo- or obligate heterodimers. Except for ABCG2,\\u000a the members of the ABCG

Hiroyuki Kusuhara; Yuichi Sugiyama



Selectivity of TMC207 towards mycobacterial ATP synthase compared with that towards the eukaryotic homologue.  


The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC(50)] of >200 microM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC(50) of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target. PMID:19075053

Haagsma, Anna C; Abdillahi-Ibrahim, Rooda; Wagner, Marijke J; Krab, Klaas; Vergauwen, Karen; Guillemont, Jerome; Andries, Koen; Lill, Holger; Koul, Anil; Bald, Dirk



Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line  

SciTech Connect

Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced /sup 86/Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. /sup 86/Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated /sup 86/Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated /sup 86/Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit /sup 86/Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes /sup 86/Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.

Steinberg, T.H.; Silverstein, S.C.



Structural Analysis of ATP Analogs Compatible with Kinase-Catalyzed Labeling  

PubMed Central

Kinase-catalyzed protein phosphorylation is an important biochemical process involved in cellular functions. We recently discovered that kinases promiscuously accept ?-modified ATP analogs as cosubstrates and used several ATP analogs as tools for studying protein phosphorylation. Herein, we explore the structural requirements of ?-modified ATP analogs for kinase compatibility. To understand the influence of linker length and composition, a series of ATP analogs was synthesized and the efficiency of kinase-catalyzed labeling was determined by quantitative mass spectrometry. This study on factors influencing kinase cosubstrate promiscuity will enable design of ATP analogs for a variety of kinase-catalyzed labeling reactions.

Suwal, Sujit; Senevirathne, Chamara; Garre, Satish; Pflum, Mary Kay H.



Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells.  


1. Increasing concentrations of ATP (0.5 microM-300 microM) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Cai responses to extracellular ATP. 2. In the absence of Mg2+ (with 3 mM CaCl2 in the buffer solution), the more sensitive response was maximal at 3-5 microM and was not further increased by 30 microM ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mM). A second, much larger increase in Cai was observed on addition of 300 microM ATP. This larger effect, which we have described previously, appears to be mediated by ATP4-, and was selectively reversed by 4,4'-di-isothiocyanato-dihydrostilbene-2,2'-disulphonate as well as by high concentrations of alpha,beta-methylene ATP. 3. Among ATP analogues, only the putative P2Z agonist, 3'-0-(4-benzoyl)benzoyl-ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP(4-)-response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP > ATP gamma S = 2-methylthio ATP (a P2y-selective agonist) > ADP, ITP and alpha,beta-methylene ATP (a P2x-selective agonist)). 4. Although the effect of ATP4- could best be characterized as a P2z-type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y-purinoceptor antagonist. Reactive blue 2 may bind to and block P2z purinoceptors since [gamma 32P]-ATP binding to parotid cells was inhibited by this compound. 5. In contrast to the response to ATP4-, the more sensitive response to ATP was potentiated by 2+ reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+.6. Parasympathetic denervation selectively increased the more sensitive response, suggesting that it maybe physiologically regulated. PMID:8448596

McMillian, M K; Soltoff, S P; Cantley, L C; Rudel, R; Talamo, B R



Overview of photo-induced therapy for ATP production  

NASA Astrophysics Data System (ADS)

The purpose of this report is to provide a review of the effects of low-power photo-induced therapy using lasers of different device parameters such as intensity, wavelength, lasing mechanism (i.e., pulsed or continuous) on the production of Adenosine triphosphate (ATP) in mammalian cells. This is a very important research topic as it is suggested in literature that there might be a relationship between the ATP levels and specific diseases. It has been shown that the ATP production was enhanced at wavelengths ranging between 600 nm and 1000 nm (also known as the optical window), in particular at 600nm, 632.8nm, 635nm, 650nm, and 904nm. However, certain experiments showed that the effectiveness of the photo-induced therapy was also dependent on the dosage and the duration of the supplied light. We present the research conclusions drawn from the experiments reported within the last decade, and provide a list of potential medical treatment(s) for patients using visible and near infrared (NIR) light.

Abdalla, Mohamed; Nagy, A.; Ye, W. N.; Mussivand, T.


Expression cloning of an ATP receptor from mouse neuroblastoma cells.  

PubMed Central

Extracellular ATP activates cell-surface metabotropic and ionotropic nucleotide (P2) receptors in vascular, neural, connective, and immune tissues. These P2 receptors mediate a wealth of physiological processes, including nitric oxide-dependent vasodilation of vascular smooth muscle and fast excitatory neurotransmission in sensory afferents. Although ATP is now recognized as a signaling molecule, the cellular and molecular mechanisms underlying its actions have been difficult to study due to the absence of selective P2 receptor antagonists and cloned receptor genes. Nonetheless, five mammalian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein-coupled peptide receptors. When expressed in Xenopus laevis oocytes, the cloned receptor resembles a metabotropic P2U receptor; activation by either ATP or UTP elicits the mobilization of intracellular calcium. mRNA encoding the P2U purinergic receptor is found in neural and nonneural tissues. Images Fig. 3 Fig. 5

Lustig, K D; Shiau, A K; Brake, A J; Julius, D



Uptake of AMP, ADP, and ATP in Escherichia coli W.  


The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-?-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-³H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-¹?C]AMP and T-1 cells. Uptake of [2-³H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells. PMID:21228488

Watanabe, Kimiko; Tomioka, Satsuki; Tanimura, Kiyoko; Oku, Hisae; Isoi, Koichiro



NASA thesaurus. Volume 3: Definitions  

NASA Technical Reports Server (NTRS)

Publication of NASA Thesaurus definitions began with Supplement 1 to the 1985 NASA Thesaurus. The definitions given here represent the complete file of over 3,200 definitions, complimented by nearly 1,000 use references. Definitions of more common or general scientific terms are given a NASA slant if one exists. Certain terms are not defined as a matter of policy: common names, chemical elements, specific models of computers, and nontechnical terms. The NASA Thesaurus predates by a number of years the systematic effort to define terms, therefore not all Thesaurus terms have been defined. Nevertheless, definitions of older terms are continually being added. The following data are provided for each entry: term in uppercase/lowercase form, definition, source, and year the term (not the definition) was added to the NASA Thesaurus. The NASA History Office is the authority for capitalization in satellite and spacecraft names. Definitions with no source given were constructed by lexicographers at the NASA Scientific and Technical Information (STI) Facility who rely on the following sources for their information: experts in the field, literature searches from the NASA STI database, and specialized references.




EPA Science Inventory

The most desirable definition of success from the point of view of environmental stewardship is to treat the entire plume to MCLs. Technically, has proven impossible to attain at many sites. A less desirable definition of success is to contain the source of contamination and tr...


Definitions: Dealing with Categories Mathematically.  

ERIC Educational Resources Information Center

Explores differences between mathematical definitions and non-mathematical definitions by contrasting three approaches to mathematical reasoning. Examines the consequences of the use of those approaches in a first course in real analysis. Analyzes students' reasoning behavior during analysis and suggests reasons for students' difficulties with the…

Alcock, Lara; Simpson, Adrian



On some definitions of mindfulness  

Microsoft Academic Search

The Buddhist technical term was first translated as ‘mindfulness’ by T.W. Rhys Davids in 1881. Since then various authors, including Rhys Davids, have attempted definitions of what precisely is meant by mindfulness. Initially these were based on readings and interpretations of ancient Buddhist texts. Beginning in the 1950s some definitions of mindfulness became more informed by the actual practice of

Rupert Gethin



Taylor Approximations and Definite Integrals  

ERIC Educational Resources Information Center

We investigate the possibility of approximating the value of a definite integral by approximating the integrand rather than using numerical methods to approximate the value of the definite integral. Particular cases considered include examples where the integral is improper, such as an elliptic integral. (Contains 4 tables and 2 figures.)

Gordon, Sheldon P.



Definitions and implications of death.  


Understanding the legal definition of whole-brain death is imperative for hematologists and oncologists who deal with end-of-life patients on a regular basis. At present, only whole-brain death in which there is no function of the upper brain or brain stem is legally recognized as legal death. Those advocating expansion of the current definition of death to encompass patients with higher brain death and brain-absent anencephalic infants cite increasing the organ pool and decreasing unnecessary treatment and costs as benefits. Those advocating a more narrow definition of death typically fear being misdiagnosed or prefer the traditional cardiopulmonary definition for personal and religious reasons. As medical technology advances, offering new hope to both the critically injured patients who might be potential donors and to those patients in need of donated organs, the definition of death will continue to be a topic of passionate debate. PMID:12512174

Schlotzhauer, Anna V; Liang, Bryan A



The enigmatic mitochondrial ORF ymf39 codes for ATP synthase chain b  

PubMed Central

ymf39 is a conserved hypothetical protein-coding gene found in mitochondrial genomes of land plants and certain protists. We speculated earlier, based on a weak sequence similarity between Ymf39 from a green alga and the atpF gene product from Bradyrhizobium, that ymf39 might code for subunit b of mitochondrial F0F1-ATP synthase. To test this hypothesis, we have sequenced ymf39 from five protists with minimally derived mitochondrial genomes, the jakobids. In addition, we isolated the mitochondrial ATP synthase complex of the jakobid Seculamonas ecuadoriensis and determined the partial protein sequence of the 19-kDa subunit, the size expected for Ymf39. The obtained peptide sequence matches perfectly with a 3?-proximal region of the ymf39 gene of this organism, confirming that Ymf39 is indeed an ATP synthase subunit. Finally, we employed statistical tests to assess the significance of sequence similarity of Ymf39 proteins with each other, their nucleus-encoded functional counterparts, ATP4/ATP5F, from fungi and animals and ?-proteobacterial ATP synthase b-subunits. This analysis provides clear evidence that ymf39 is an atpF homolog, while ATP4/ATP5F appears to be a highly diverged form of ymf39 that has migrated to the nucleus. We propose to designate ymf39 from now on atp4.

Burger, Gertraud; Lang, B. Franz; Braun, Hans-Peter; Marx, Stefanie



Photoaffinity labeling of rat liver carbamoyl phosphate synthetase I by 8-azido-ATP  

SciTech Connect

8-Azido-ATP has been found to serve as a photoaffinity label for two distinct ATP sites on rat liver carbamoyl phosphate synthetase I and to allow preliminary localization of these sites. In the dark, 8-azido-ATP acted as a competitive inhibitor with respect to ATP. Ultraviolet irradiation of carbamoyl phosphate synthetase I in the presence of 8-azido-ATP led to an irreversible loss of activity. ATP specifically protected against this inactivation. The incorporation of 2 mol of 8-azido-ATP per mol of enzyme was required for complete inactivation. To localize the 8-azido-ATP-binding sites to discrete regions of carbamoyl phosphate synthetase I which appear to be structural domains, the enzyme was photolabeled with (gamma-/sup 32/P)8-azido-ATP and subjected to limited proteolytic digestion. The resulting model for the functional roles of the domains is that there is one ATP site on each of the two large internal structural domains of the enzyme. Each of these domains was found to contain the consensus sequences A and B common to many other nucleotide-binding proteins. In addition, there is extensive structural and possibly functional interaction of the smaller N-terminal domain with one of the internal ATP-binding domains, analogous to a subunit interaction observed with the evolutionarily related Escherichia coli carbamoyl phosphate synthetase.

Powers-Lee, S.G.; Corina, K.



Multiple ATP-dependent steps in RNA polymerase II promoter melting and initiation.  

PubMed Central

Permanganate probing and abortive initiation assays were used to investigate the role of ATP in several successive stages of transcription initiation at the activated adeno E4 and mouse DHFR promoters. Removal of ATP at several points along the multi-step pathway blocked further progress towards its completion. Most strikingly, even if the DNA transcription start site is opened using ATP, the subsequent removal of ATP disallows formation of the first phosphodiester bond of the RNA. After ATP-dependent formation of a short RNA, a new transcription complex forms, which is more stable and has a longer open region. Both RNA and ATP appear to play roles in the formation of this complex. The need for ATP throughout this multi-step initiation pathway leads to new and unexpected possibilities for the use of energy and ATPases in transcription initiation.

Yan, M; Gralla, J D



Aminoacyl-tRNA synthetases: affinity labeling of the ATP binding site by 2', 3' -ribose oxidized ATP.  

PubMed Central

Homogeneous Escherichia coli methionyl-, isoleucyl-, tryptophanyl-, and phenylalanyl-tRNA synthetases and Bacillus stearothermophilus methionyl- and tyrosyl-tRNA synthetases are irreversibly inactivated by reaction of their active ATP sites with the 2',3'-dialdehyde derivative of ATP obtained by periodate oxidation. In each case, the amount of 14C-labeled dialdehyde derivative incorporated per molecule of inactivated enzyme appears consistent with the expected active stoichiometry of the synthetase. These results strongly support the presence, at the active site of the studied aminoacyl-tRNA synthetases, of a common residue, probably a lysine whose epsilon-NH2 group is known, from the work of others, to form a Schiff's base specifically with the 2',3'-dialdehyde derivatives of ribonucleotides.

Fayat, G; Fromant, M; Blanquet, S



Cervical anterior transpedicular screw fixation (ATPS)—Part II. Accuracy of manual insertion and pull-out strength of ATPS  

Microsoft Academic Search

Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or\\u000a infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these\\u000a cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular\\u000a screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications

Heiko Koller; Frank Acosta; Mark Tauber; Michael Fox; Hudelmaier Martin; Rosmarie Forstner; Peter Augat; Rainer Penzkofer; Christian Pirich; H. Kässmann; Herbert Resch; Wolfgang Hitzl



Evidence for myocardial ATP compartmentation from NMR inversion transfer analysis of creatine kinase fluxes.  

PubMed Central

The interpretation of creatine kinase (CK) flux measured by (31)P NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P(i) exchange on the forward (F(f): PCr --> ATP) and reverse (F(r)) CK fluxes derived from complete analysis of inversion transfer. Although F(f) should equal F(r) because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P(i) was masked by saturation of P(i) (sat-P(i)), F(f)/F(r) significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F(f)/F(r) for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P(i) might not result only from the masking of ATP-P(i) exchange.

Joubert, F; Gillet, B; Mazet, J L; Mateo, P; Beloeil, J; Hoerter, J A



Selective and ATP-driven transport of ions across supported membranes into nanoporous carriers using gramicidin A and ATP synthase.  


We report a robust and versatile membrane protein based system for selective uptake and release of ions from nanoporous particles sealed with ion-tight lipid bilayers of various compositions that is driven by the addition of ATP or a chemical potential gradient. We have successfully incorporated both a passive ion channel-type peptide (gramicidin A) and a more complex primary sodium ion transporter (ATP synthase) into the supported lipid bilayers on solid nanoporous silica particles. Protein-mediated controlled release/uptake of sodium ions across the ion-tight lipid bilayer seal from or into the nanoporous silica carrier was imaged in real time using a confocal laser scanning microscope and the intensity changes were quantified. ATP-driven transport of sodium ions across the supported lipid bilayer against a chemical gradient was demonstrated. The possibility of designing durable carriers with tight lipid membranes, containing membrane proteins for selective ion uptake and release, offers new possibilities for functional studies of single or cascading membrane protein systems and could also be used as biomimetic microreactors for controlled synthesis of inorganic multicomponent materials. PMID:23321853

Oliynyk, Vitaliy; Mille, Christian; Ng, Jovice B S; von Ballmoos, Christoph; Corkery, Robert W; Bergström, Lennart



Contributions of citrate in redox potential maintenance and ATP production: metabolic pathways and their regulation in Lactobacillus panis PM1.  


Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli and can utilize various NADH-reoxidizing routes (e.g., citrate, glycerol, and oxygen) according to environmental conditions. In this study, we investigated the ability of L. panis PM1 to produce succinate, acetate, and lactate via citrate utilization. Possible pathways, as well as regulation, for citrate metabolism were examined on the basis of the genome sequence data and metabolic profiles of L. panis PM1. The presence of citrate led to the up-regulation, at the transcriptional level, of the genes encoding for citrate lyase, malate dehydrogenase, and malic enzyme of the citrate pathways by 10- to 120-fold. The transcriptional regulator of the dha operon coding for glycerol dehydratase of L. panis PM1 repressed the expression of the citrate lyase gene (10-fold). Metabolite analyses indicated that the transcriptional enhancement by citrate stimulated succinate yield. Citrate metabolism contributed to energy production by providing a major alternate pathway for NAD(+) regeneration and allowed acetyl phosphate to yield acetate/ATP instead of ethanol/NAD(+). Additionally, a branching pathway from oxaloacetate to pyruvate increased the pool of lactate, which was then used to produce ATP during stationary phase. However, the redirection of NADH-to-citrate utilization resulted in stress caused by end-products (i.e., succinate and acetate). This stress reduced succinate production by up to 50 % but did not cause significant changes at transcriptional level. Overall, citrate utilization was beneficial for the growth of L. panis PM1 by providing a NAD(+) regeneration route and producing extra ATP. PMID:23912115

Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji



High definition systems in Japan  

NASA Technical Reports Server (NTRS)

The successful implementation of a strategy to produce high-definition systems within the Japanese economy will favorably affect the fundamental competitiveness of Japan relative to the rest of the world. The development of an infrastructure necessary to support high-definition products and systems in that country involves major commitments of engineering resources, plants and equipment, educational programs and funding. The results of these efforts appear to affect virtually every aspect of the Japanese industrial complex. The results of assessments of the current progress of Japan toward the development of high-definition products and systems are presented. The assessments are based on the findings of a panel of U.S. experts made up of individuals from U.S. academia and industry, and derived from a study of the Japanese literature combined with visits to the primary relevant industrial laboratories and development agencies in Japan. Specific coverage includes an evaluation of progress in R&D for high-definition television (HDTV) displays that are evolving in Japan; high-definition standards and equipment development; Japanese intentions for the use of HDTV; economic evaluation of Japan's public policy initiatives in support of high-definition systems; management analysis of Japan's strategy of leverage with respect to high-definition products and systems.

Elkus, Richard J., Jr.; Cohen, Robert B.; Dayton, Birney D.; Messerschmitt, David G.; Schreiber, William F.; Tannas, Lawrence E., Jr.; Shelton, Duane



Supramolecular interaction between water-soluble cali[4]xarene and ATP—the catalysis of calix[4]arene for hydrolysis of ATP  

Microsoft Academic Search

The catalysis effect of water-soluble calix[4]arene C[4] (calix[4]arene-5,11,17,23-tetrasulfonate) on hydrolysis of ATP in aqueous solution was studied by HPLC. Using laser photolysis and pulse radiolysis, the supramolecular interaction between water-soluble calix[4]arene and ATP was investigated.

Yao Tian-Ming; Ye Zhi-Feng; Wang Li; Gu Jin-Ying; Yao Si-De; Shi Xian-Fa



Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability.  


The ability of Mycobacterium tuberculosis to persist in its human host despite extensive chemotherapy is thought to be based on subpopulations of non-replicating phenotypically drug-resistant bacilli. To study the non-growing pathogen, culture models that generate quiescent organisms by either oxygen depletion in nutrient-rich medium (Wayne model) or nutrient deprivation in oxygen-rich medium (Loebel model) have been developed. In contrast to the energy metabolism of Wayne bacilli, little is known about Loebel bacilli. Here we analysed M. tuberculosis under nutrient-starvation conditions. Upon shifting to the non-replicating state the pathogen maintained a fivefold reduced but constant intracellular ATP level. Chemical probing of the F(0)F(1) ATP synthase demonstrated the importance of this enzyme for ATP homeostasis and viability of the nutrient-starved organism. Surprisingly, the specific ATP synthase inhibitor TMC207 did not affect viability and only moderately reduced the intracellular ATP level of nutrient-starved organisms. Depletion of oxygen killed Loebel bacilli, whereas death was prevented by nitrate, suggesting that respiration and an exogenous electron acceptor are required for maintaining viability. Nutrient-starved bacilli lacking the glyoxylate shunt enzyme isocitrate lyase failed to reduce their intracellular ATP level and died, thus establishing a link between ATP control and intermediary metabolism. We conclude that reduction of the ATP level might be an important step in the adaptation of M. tuberculosis to non-growing survival. PMID:19797356

Gengenbacher, Martin; Rao, Srinivasa P S; Pethe, Kevin; Dick, Thomas



Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold.  


The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix ?inh that is provided by a cognate anti-toxin (class I), or is part of the N- or C-terminal part of the Fic protein itself (classes II and III). In vitro, inhibition is relieved by mutation of a conserved glutamate of ?inh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the ?-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The ?-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an ?-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain ?-interactions with the ?-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein. PMID:23738009

Goepfert, Arnaud; Stanger, Frédéric V; Dehio, Christoph; Schirmer, Tilman



Bifidobacterium lactis DSM 10140: Identification of the atp (atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny  

PubMed Central

The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F1F0-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.

Ventura, Marco; Canchaya, Carlos; van Sinderen, Douwe; Fitzgerald, Gerald F.; Zink, Ralf



ATP Produces Vasodilation via P1 Purinoceptors and Vasoconstriction via P2 Purinoceptors in the Isolated Rabbit Central Ear Artery  

Microsoft Academic Search

P1 and P2 purinoceptors mediating mechanical responses in isolated rabbit ear artery were studied by comparing responses to adenosine triphosphate (ATP), ??-methylene ATP, and adenosine, both when endothelial cells were intact and when they had been removed by mechanical rubbing (as confirmed by histochemical staining and near abolition of relaxation to acetylcholine). ??-Methylene ATP and ATP (but not adenosine or

Charles Kennedy; Geoffrey Burnstock



Sopite syndrome: a revised definition.  


In 1976, Graybiel and Knepton proposed the term "sopite syndrome" to describe a symptom complex centering on drowsiness and lethargy related to motion sickness. However, existing descriptions and definitions of sopite syndrome have limitations in fully conveying the appropriate information to the reader. Our objective is to propose a revised definition providing a more adequate conceptual framework for research. The proposed definition of sopite syndrome addresses the nonspecificity of soporific symptoms, the health state of the individuals, and the existence of a motion stimulus. PMID:24919391

Matsangas, Panagiotis; McCauley, Michael E



Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor.  


R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDR Mycobacterium tuberculosis isolates. In the present study, we characterized the development of resistance to R207910 in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates of M. tuberculosis (three drug-susceptible and four MDR isolates) at 10x, 30x, and 100x the MIC. At a concentration of 0.3 mg/liter (10x the MIC), the mutation rates ranged from 4.7 x 10(-7) to 8.9 x 10(-9) mutations per cell per division, and at 1.0 mg/liter (30x the MIC) the mutation rate ranged from 3.9 x 10(-8) to 2.4 x 10(-9). No resistant mutants were obtained at 3 mg/liter (100x the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, the atpE gene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in the atpE gene. For 38/53 mutants, no atpE mutations were found and sequencing of the complete F0 ATP synthase operon (atpB, atpE, and atpF genes) and the F1 ATP synthase operon (atpH, atpA, atpG, atpD, and atpC genes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types. PMID:20038615

Huitric, E; Verhasselt, P; Koul, A; Andries, K; Hoffner, S; Andersson, D I



Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).  


A soluble ATP-diphosphohydrolase (apyrase, EC has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (apyrase conserved regions) 1-4 that are highly conserved in known apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed. PMID:8579614

Handa, M; Guidotti, G



Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone  

PubMed Central

Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATP?S reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.

Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan; Sengupta, Rituparna; Jiang, Li; Satyshur, Kenneth A; Xing, Yongna



ATP Antagonizes Thrombin-Induced Signal Transduction through 12(S)-HETE and cAMP  

PubMed Central

In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca2+]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists.

Burzaco, Jaione; Conde, Manuel; Parada, Luis A.; Zugaza, Jose L.; Dehaye, Jean-Paul; Marino, Aida



Wound-induced ATP release and EGF receptor activation in epithelial cells  

PubMed Central

Summary We have shown previously that wounding of human corneal epithelial (HCE) cells resulted in epidermal growth factor receptor (EGFR) transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the initial signal to trigger these signaling events in response to cell injury remains elusive. In the present study, we investigated the role of ATP released from the injured cells in EGFR transactivation in HCE cells as well as in BEAS 2B cells, a bronchial epithelial cell line. Wounding of epithelial monolayer resulted in the release of ATP into the culture medium. The wound-induced rapid activation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways in HCE cells was attenuated by eliminating extracellular ATP, ADP and adenosine. The nonhydrolyzable ATP analog ATP-?-S induced rapid and sustained EGFR activation that depended on HB-EGF shedding and ADAM (a disintegrin and metalloproteinase). Targeting pathways leading to HB-EGF shedding and EGFR activation attenuated ATP-?-S-enhanced closure of small scratch wounds. The purinoceptor antagonist reactive blue 2 decreased wound closure and attenuated ATP-?-S induced HB-EGF shedding. Taken together, our data suggest that ATP, released upon epithelial injury, acts as an early signal to trigger cell responses including an increase in HB-EGF shedding, subsequent EGFR transactivation and its downstream signaling, resulting in wound healing.

Yin, Jia; Xu, Keping; Zhang, Jing; Kumar, Ashok; Yu, Fu-Shin X.



How Reliable Are ATP Bioluminescence Meters in Assessing Decontamination of Environmental Surfaces in Healthcare Settings?  

PubMed Central

Background Meters based on adenosine triphosphate (ATP) bioluminescence measurements in relative light units (RLU) are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? Objective This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals’ interference in their readings. Methods First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter’s ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings. Results All four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated. Conclusions Apart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries.

Omidbakhsh, Navid; Ahmadpour, Faraz; Kenny, Nicole



Adenosine-5'-Triphosphate (ATP) Protects Mice against Bacterial Infection by Activation of the NLRP3 Inflammasome  

PubMed Central

It has been established that Adenosine-5'-triphosphate (ATP) can activate the NLRP3 inflammasome. However, the physiological effect of extracellular ATP on NLRP3 inflammasome activation has not yet been investigated. In the present study, we found that ATP was indeed released during bacterial infection. By using a murine peritonitis model, we also found that ATP promotes the fight against bacterial infection in mice. ATP induced the secretion of IL-1? and chemokines by murine bone marrow-derived macrophages in vitro. Furthermore, the intraperitoneal injection of ATP elevated the levels of IL-1? and chemokines in the mouse peritoneal lavage. Neutrophils were rapidly recruited to the peritoneum after ATP injection. In addition, the effects on cytokine and chemokine secretion and neutrophil recruitment were markedly attenuated by the pre-administration of the caspase-1 inhibitor Ac-YVAD-cho. Ac-YVAD-cho also significantly attenuated the protective effect of ATP against bacterial infection. In the present study, we demonstrated a protective role for ATP during bacterial infection and this effect was related to NLRP3 inflammasome activation. Together, these results suggest a role for ATP in initiating the immune response in hosts suffering from infections.

Yan, Chao; Gao, Qian; Li, Sheng-An; Liu, Jie; Zhou, Kaifeng; Guo, Xiaolong; Lee, Wenhui; Zhang, Yun



Modeling the effects of hypoxia on ATP turnover in exercising muscle  

NASA Technical Reports Server (NTRS)

Most models of metabolic control concentrate on the regulation of ATP production and largely ignore the regulation of ATP demand. We describe a model, based on the results of Hogan et al. (J. Appl. Physiol. 73: 728-736, 1992), that incorporates the effects of ATP demand. The model is developed from the premise that a unique set of intracellular conditions can be measured at each level of ATP turnover and that this relationship is best described by energetic state. Current concepts suggest that cells are capable of maintaining oxygen consumption in the face of declines in the concentration of oxygen through compensatory changes in cellular metabolites. We show that these compensatory changes can cause significant declines in ATP demand and result in a decline in oxygen consumption and ATP turnover. Furthermore we find that hypoxia does not directly affect the rate of anaerobic ATP synthesis and associated lactate production. Rather, lactate production appears to be related to energetic state, whatever the PO2. The model is used to describe the interaction between ATP demand and ATP supply in determining final ATP turnover.

Arthur, P. G.; Hogan, M. C.; Bebout, D. E.; Wagner, P. D.; Hochachka, P. W.



Mitochondrial Calcium Signaling Mediates Rhythmic Extracellular ATP Accumulation in Suprachiasmatic Nucleus Astrocytes  

PubMed Central

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of adenosine triphosphate (ATP). Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Since ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca2+ and ATP accumulation and have demonstrated that intracellular Ca2+ levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca2+ levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca2+. In contrast, our finding that peak mitochondrial Ca2+ occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca2+uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Taken together, these results demonstrate that mitochondrial Ca2+mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

Burkeen, Jeff F.; Womac, Alisa D.; Earnest, David J.; Zoran, Mark J.



Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP  

SciTech Connect

The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes /sup 14/C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg/sup 2 +/. Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-..gamma..-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated.

Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.



Intracellular ATP binding is required to activate the slowly activating K+ channel IKs  

PubMed Central

Gating of ion channels by ligands is fundamental to cellular function, and ATP serves as both an energy source and a signaling molecule that modulates ion channel and transporter functions. The slowly activating K+ channel IKs in cardiac myocytes is formed by KCNQ1 and KCNE1 subunits that conduct K+ to repolarize the action potential. Here we show that intracellular ATP activates heterologously coexpressed KCNQ1 and KCNE1 as well as IKs in cardiac myocytes by directly binding to the C terminus of KCNQ1 to allow the pore to open. The channel is most sensitive to ATP near its physiological concentration, and lowering ATP concentration in cardiac myocytes results in IKs reduction and action potential prolongation. Multiple mutations that suppress IKs by decreasing the ATP sensitivity of the channel are associated with the long QT (interval between the Q and T waves in electrocardiogram) syndrome that predisposes afflicted individuals to cardiac arrhythmia and sudden death. A cluster of basic and aromatic residues that may form a unique ATP binding site are identified; ATP activation of the wild-type channel and the effects of the mutations on ATP sensitivity are consistent with an allosteric mechanism. These results demonstrate the activation of an ion channel by intracellular ATP binding, and ATP-dependent gating allows IKs to couple myocyte energy state to its electrophysiology in physiologic and pathologic conditions.

Li, Yang; Gao, Junyuan; Lu, Zhongju; McFarland, Kelli; Shi, Jingyi; Bock, Kevin; Cohen, Ira S.; Cui, Jianmin



Ligand-dependent linkage of the ATP site to inhibition gate closure in the KATP channel.  


Major advances have been made on the inhibition gate and ATP site of the K(ir)6.2 subunit of the K(ATP) channel, but little is known about conformational coupling between the two. ATP site mutations dramatically disrupt ATP-dependent gating without effect on ligand-independent gating, observed as interconversions between active burst and inactive interburst conformations in the absence of ATP. This suggests that linkage between site and gate is conditionally dependent on ATP occupancy. We studied all substitutions at position 334 of the ATP site in K(ir)6.2deltaC26 that express in Xenopus oocytes. All substitutions disrupted ATP-dependent gating by 10-fold or more. Only positive-charged arginine or lysine at 334, however, slowed ligand-independent gating from the burst, and this was in some but not all patches. Moreover, the polycationic peptide protamine reversed the slowed gating from the burst of 334R mutant channels, and speeded the slow gating from the burst of wild-type SUR1/K(ir)6.2 in the absence of ATP. Our results support a two-step ligand-dependent linkage mechanism for K(ir)6.2 channels in which ATP-occupied sites function to electrostatically dissociate COOH-terminal domains from the membrane, then as in all K(ir) channels, free COOH-terminal domains and inner M2 helices transit to a lower energy state for gate closure. PMID:16129775

Li, Lehong; Geng, Xuehui; Yonkunas, Michael; Su, Anjey; Densmore, Erik; Tang, Pei; Drain, Peter



ATP induces conformational changes in the carboxyl-terminal region of ClC-5.  


ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation. PMID:21173145

Wellhauser, Leigh; Luna-Chavez, Cesar; D'Antonio, Christina; Tainer, John; Bear, Christine E



ATP Induces Conformational Changes in the Carboxyl-terminal Region of ClC-5*  

PubMed Central

ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation.

Wellhauser, Leigh; Luna-Chavez, Cesar; D'Antonio, Christina; Tainer, John; Bear, Christine E.



Identification of residues contributing to the ATP binding site of Kir6.2  

PubMed Central

The ATP-sensitive potassium (KATP) channel links cell metabolism to membrane excitability. Intracellular ATP inhibits channel activity by binding to the Kir6.2 subunit of the channel, but the ATP binding site is unknown. Using cysteine-scanning mutagenesis and charged thiol-modifying reagents, we identified two amino acids in Kir6.2 that appear to interact directly with ATP: R50 in the N-terminus, and K185 in the C-terminus. The ATP sensitivity of the R50C and K185C mutant channels was increased by a positively charged thiol reagent (MTSEA), and was reduced by the negatively charged reagent MTSES. Comparison of the inhibitory effects of ATP, ADP and AMP after thiol modification suggests that K185 interacts primarily with the ?-phosphate, and R50 with the ?-phosphate, of ATP. A molecular model of the C-terminus of Kir6.2 (based on the crystal structure of Kir3.1) was constructed and automated docking was used to identify residues interacting with ATP. These results support the idea that K185 interacts with the ?-phosphate of ATP. Thus both N- and C-termini may contribute to the ATP binding site.

Trapp, Stefan; Haider, Shozeb; Jones, Phillippa; Sansom, Mark S.P.; Ashcroft, Frances M.



50 CFR 17.3 - Definitions.  

Code of Federal Regulations, 2010 CFR

...Provisions § 17.3 Definitions. In addition to the definitions contained in part 10 of...culling, contraception, euthanasia, grouping or handling...endangered. Harass in the definition of âtakeâ in the...



50 CFR 17.3 - Definitions.  

Code of Federal Regulations, 2010 CFR

...Provisions § 17.3 Definitions. In addition to the definitions contained in part 10 of...culling, contraception, euthanasia, grouping or handling...endangered. Harass in the definition of âtakeâ in the...



41 CFR 60-3.16 - Definitions.  

Code of Federal Regulations, 2010 CFR

...PROCEDURES (1978) Definitions § 60-3.16 Definitions. The following definitions shall apply throughout... Demonstrated by empirical data showing that the...responsibilities. M. Knowledge. A body of...



76 FR 53376 - Definition of Solid Waste  

Federal Register 2010, 2011, 2012, 2013

...RIN 2050-AG62 Definition of Solid Waste AGENCY: Environmental Protection...proposed rule on the definition of solid waste published in the Federal Register...exclusions from the definition of solid waste for hazardous secondary...



49 CFR 1101.1 - Statutory definitions.  

Code of Federal Regulations, 2013 CFR

...Transportation (Continued) SURFACE TRANSPORTATION BOARD, DEPARTMENT OF TRANSPORTATION RULES OF PRACTICE DEFINITIONS AND CONSTRUCTION § 1101.1 Statutory definitions. The definitions contained in section 10102 of the Act (49...



48 CFR 2002.100 - Definitions.  

Code of Federal Regulations, 2013 CFR

...Section 2002.100 Federal Acquisition Regulations System NUCLEAR REGULATORY COMMISSION GENERAL DEFINITIONS Definitions 2002.100 Definitions. Agency means the Nuclear Regulatory Commission (NRC). Agency Head or Head...



76 FR 80846 - Definition of Enforcement Action  

Federal Register 2010, 2011, 2012, 2013

...Part 502 RIN 3141-AA43 Definition of Enforcement Action AGENCY: National Indian Gaming...regulations to include definitions for ``enforcement action''. The Indian Gaming Regulatory...them. Therefore, a definition of ``enforcement action'' is proposed in this...



Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions  

PubMed Central

The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca2+ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist ?,?-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M2-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M2-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca2+ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies.

Sui, Guiping; Fry, Chris H.; Montgomery, Bruce; Roberts, Max; Wu, Rui



Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis  

PubMed Central

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1's expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.

Feng, Shengmei; Zhu, Guochun; McConnell, Matthew; Deng, Lianfu; Zhao, Qiang; Wu, Mengrui; Zhou, Qi; Wang, Jinshen; Qi, Jin; Li, Yi-Ping; Chen, Wei



Mitogenic effects of ATP on vascular smooth muscle cells vs. other growth factors and sympathetic cotransmitters.  


The sympathetic nervous system has been shown to exert a trophic influence on vascular smooth muscle cells (VSMC). Therefore, we studied the growth-regulating effects of the sympathetic cotransmitters ATP, neuropeptide Y (NPY), and norepinephrine (NE). ATP in concentrations of 1-100 microM greatly increased the incorporation of [3H]thymidine in VSMC from rat aorta and vena cava. ATP also increased cell number and total protein content. The maximal effect on [3H]thymidine incorporation was greater than for epidermal growth factor (20 ng/ml) or insulin (1 microgram/ml) and approximately one-half that of 10% fetal calf serum. The potency series of other nucleotides and analogues of ATP was ATP > beta, gamma-methyleneATP (AMP-PCP) > ADP > adenosine > alpha, beta- methyleneATP (AMP-CPP) > 2-methylthioATP, indicating involvement of a P2 receptor, however, it does not meet proposed pharmacological criteria of either the P2x or P2y subclass. Several proposed P2 receptor antagonists were without effect. The effect of ATP could be mediated by a "nucleotide receptor," since UTP also stimulated [3H]thymidine incorporation. In our model, there was a strong correlation between the mitogenic effects of ATP, AMP-CPP, AMP-PCP, and UTP and their ability to stimulate influx of extracellular Ca2+ (Ca2+o). Moreover, the mitogenic effect of ATP was increased by high concentrations of Ca2+o. Taken together with data showing the lack of involvement of several other second-messenger systems, this indicates a critical role for Ca2+o in mediating the mitogenic effects of ATP. Amiloride, known to inhibit the action of several growth factors, also inhibited ATP-induced mitogenesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7694483

Erlinge, D; Yoo, H; Edvinsson, L; Reis, D J; Wahlestedt, C



Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures  

PubMed Central

ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential (??m; TMRM) and NAD(P)H autofluorescence. In embryonic neurons, transient glucose deprivation induced a near-complete decrease in [ATP]c, which was partially reversible and was accelerated by inhibition of glycolysis with 2-deoxyglucose. In the absence of glucose, pyruvate did not cause any significant increase in [ATP]c in 84% of embryonic neurons, and inhibition of mitochondrial ATP synthase with oligomycin failed to decrease [ATP]c. Moreover, ??m was significantly reduced by oligomycin, indicating that mitochondria acted as consumers rather than producers of ATP in embryonic neurons. In sharp contrast, in postnatal neurons pyruvate added during glucose deprivation significantly increased [ATP]c (by 54 ± 8%), whereas oligomycin induced a sharp decline in [ATP]c and increased ??m. These signs of oxidative phosphorylation were observed in all tested P1–P2 neurons. Measurement of ??m with the potential-sensitive probe JC-1 revealed that neuronal mitochondrial membrane potential was significantly reduced in embryonic cultures compared to the postnatal ones, possibly due to increased proton permeability of inner mitochondrial membrane. We conclude that, in embryonic, but not postnatal neuronal cultures, ATP synthesis is predominantly glycolytic and the oxidative phosphorylation-mediated synthesis of ATP by mitochondrial F1Fo-ATPase is insignificant.

Surin, Alexander M.; Khiroug, Serguei; Gorbacheva, Lubov R.; Khodorov, Boris I.; Pinelis, Vsevolod G.; Khiroug, Leonard



ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces.  


A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria and Planococcus secreted up to 4.1 pM ATP/cfu. Variations in the levels of extracellular and intracellular ATP-dependent luminescence were monitored in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (poly[tert-butyl methacrylate], PtBMA) and hydrophilic (mica) surfaces. The bacteria responded to different polymeric surfaces by producing either intracellular or extracellular ATP. The level of intracellular ATP in S. mediterraneus ATCC 700856T attached to either surface was as high as 50-55 pM ATP/cfu, while in P. maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively. S. mediterraneus ATCC 700856T generated about 20 and 50 pM of extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated by P. maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu. The levels of extracellular ATP generated by S. mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution atomic force microscopy imaging revealed a potentially interesting correlation between the porous cell-surface of certain alpha- and gamma-proteobacteria and their ability to secrete high amounts of ATP. PMID:16636988

Ivanova, Elena P; Alexeeva, Yulia V; Pham, Duy K; Wright, Jonathan P; Nicolau, Dan V




ERIC Educational Resources Information Center

SUPERSTARS III is a K-8 program designed as an enrichment opportunity for self-directed learners in mathematics. The basic purpose of SUPERSTARS III is to provide the extra challenge that self-motivated students need in mathematics and to do so in a structured, long-term program that does not impinge on the normal classroom routine or the…

North Carolina State Dept. of Public Education, Raleigh.


Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme.  


Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range. The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases. PMID:2530585

Kasho, V N; Boyer, P D



Quality medical care. A definition.  


This article offers a definition of quality medical care. Quality itself is defined not as consisting of the properties of an object but rather as the capacity of these properties to achieve goals. Accordingly, quality medical care is the capacity of the elements of that care to achieve legitimate medical and nonmedical goals. This definition is compared with other current definitions. I offer answers to the questions of how to choose goals, who chooses goals, and what are legitimate goals. Implications of this definition are discussed, particularly with reference to chart review. Because patient values shape goals and because these values are not always assessed and recorded, it is recommended that a formal assessment of patient values become part of the patient's record. PMID:3379723

Steffen, G E



PEP solar array definition study  

NASA Technical Reports Server (NTRS)

The conceptual design of a large, flexible, lightweight solar array is presented focusing on a solar array overview assessment, solar array blanket definition, structural-mechanical systems definition, and launch/reentry blanket protection features. The overview assessment includes a requirements and constraints review, the thermal environment assessment on the design selection, an evaluation of blanket integration sequence, a conceptual blanket/harness design, and a hot spot analysis considering the effects of shadowing and cell failures on overall array reliability. The solar array blanket definition includes the substrate design, hinge designs and blanket/harness flexibility assessment. The structural/mechanical systems definition includes an overall loads and deflection assessment, a frequency analysis of the deployed assembly, a components weights estimate, design of the blanket housing and tensioning mechanism. The launch/reentry blanket protection task includes assessment of solar cell/cover glass cushioning concepts during ascent and reentry flight condition.



NCI-Frederick PHL - Definitions

Services Price List Courier Services & Shipment Procedures Scheduling Contact Information Related Links Establishing an Account PHL Forms PHL Portal Definitions Antibody development:Optimize the techniques for the visualization of a new antibody. Bioassay/phenotyping


TRWG Definition of Translational Research

TRWG Definition of Translational Research The Translational Research Working Group (TRWG) defines Translational Research in the following way: "Translational research transforms scientific discoveries arising from laboratory, clinical, or population studies


Changing definitions of metabolic syndrome  

PubMed Central

The first description of patients with clustering of various metabolic abnormalities was as early as 1923 but it was more than five decades later, in 1988, that Reaven coined the term ‘syndrome X’ for this entity. The last two decades have brought forth a number of definitions and criteria to identify this condition. Various studies have demonstrated disparities in these definitions and a few researchers have questioned the utility of these criteria and even the existence of such a syndrome. A few important definitions are reviewed in this paper and, at the end, a simplified clinical definition is given and a simple parameter – lipid accumulation product – is been described that can be used to identify this condition.

Parikh, Rakesh M.; Mohan, Viswanathan



Report of the Adult Treatment Panel III: the 2001 National Cholesterol Education Program guidelines on the detection, evaluation and treatment of elevated cholesterol in adults.  


The ATP III report represents an important advance from previous ATP reports dating back to the late 1980s. The guidelines are more tightly evidence-based than previous reports, partly because of evolution of the guideline process, requiring clearly delineated links between evidence and recommendations and also because of the robust evidence base published over the last decade. An important change in ATP III is the expansion of the high-risk category to include patients without evident vascular disease, but with a level of risk equivalent to those patients with established CHD. This group termed "coronary heart disease equivalents" now includes patients with diabetes, and those with a 10-year absolute risk of over 20 percent for CHD events. With the ATP III report, the Framingham risk score is formally introduced into the guideline process. The scoring system allows for easy calculation of the absolute risk for an individual of having a "hard" CHD event (myocardial infarction, or CHD death). The report also discusses in detail concepts of lifetime or long-term risk. ATP III has broadened recommendations for lifestyle change termed "therapeutic lifestyle change (TLC)," and eliminated the step 1 and step 2 diet approach. Finally, the report details established approaches to improve adherence and provides patients and clinicians with a set of implementation tools to enhance use of the guidelines and compliance with the guidelines' recommendations. It is hoped that by improved understanding, recognition of a firm evidence base, and education through multiple channels, that adherence with the new ATP III guidelines will improve the care of our population by more effectively targeting lipid factors that lead to the development and progression of atherosclerotic cardiovascular disease. PMID:14621453

Pasternak, Richard C



Definition and Quantitation in Ecology  

Microsoft Academic Search

AUSTIN1, in commenting on my communication2, has provided an example of the problems which arise in justifying a non-quantitative approach to a subject such as ecology. Austin attaches significance to an imprecision of definition in my examination of the relationships among functional properties of Californian grasslands, and concludes ``more adequate definitions are necessary before McNaughton's suggestions ... can profitably be

S. J. McNaughton



Nutraceutical-definition and introduction  

Microsoft Academic Search

Dr Stephen DeFelice coined the term “Nutraceutical” from “Nutrition” and “Pharmaceutical” in 1989. The term nutraceutical\\u000a is being commonly used in marketing but has no regulatory definition. An attempt to redefine nutraceuticals and functional\\u000a foods is made in this article. The proposed definitions can help distinguish between functional foods, nutraceuticals, and\\u000a dietary supplements. The advantages and disadvantages of nutraceuticals are

Ekta K. Kalra



Cyano-bridged Mn(III)-M(III) single-chain magnets with M(III)=Co(III), Fe(III), Mn(III), and Cr(III).  


A series of isostructural cyano-bridged Mn(III)(h.s.)-M(III)(l.s.) alternating chains, [Mn(III)(5-TMAMsalen)M(III)(CN)(6)]?4H(2)O (5-TMAMsalen(2-)=N,N'-ethylenebis(5-trimethylammoniomethylsalicylideneiminate), Mn(III)(h.s.)=high-spin Mn(III), M(III)(l.s.)=low-spin Co(III), Mn-Co; Fe(III), Mn-Fe; Mn(III), Mn-Mn; Cr(III), Mn-Cr) was synthesized by assembling [Mn(III)(5-TMAMsalen)](3+) and [M(III)(CN)(6)](3-). The chains present in the four compounds, which crystallize in the monoclinic space group C2/c, are composed of an [-Mn(III)-NC-M(III)-CN-] repeating motif, for which the -NC-M(III)-CN- motif is provided by the [M(III)(CN)(6)](3-) moiety adopting a trans bridging mode between [Mn(III)(5-TMAMsalen)](3+) cations. The Mn(III) and M(III) ions occupy special crystallographic positions: a C(2) axis and an inversion center, respectively, forming a highly symmetrical chain with only one kind of cyano bridge. The Jahn-Teller axis of the Mn(III)(h.s.) ion is perpendicular to the N(2)O(2) plane formed by the 5-TMAMsalen tetradentate ligand. These Jahn-Teller axes are all perfectly aligned along the unique chain direction without a bending angle, although the chains are corrugated with an Mn-N(axis) -C angle of about 144°. In the crystal structures, the chains are well separated with the nearest inter-chain M???M distance being relatively large at 9?Å due to steric hindrance of the bulky trimethylammoniomethyl groups of the 5-TMAMsalen ligand. The magnetic properties of these compounds have been thoroughly studied. Mn-Fe and Mn-Mn display intra-chain ferromagnetic interactions, whereas Mn-Cr is characterized by an antiferromagnetic exchange that induces a ferrimagnetic spin arrangement along the chain. Detailed analyses of both static and dynamic magnetic properties have demonstrated without ambiguity the single-chain magnet (SCM) behavior of these three systems, whereas Mn-Co is merely paramagnetic with S(Mn)=2 and D/k(B)=-5.3?K (D being a zero-field splitting parameter). At low temperatures, the Mn-M compounds with M=Fe, Mn, and Cr display remarkably large M versus H hysteresis loops for applied magnetic fields along the easy magnetic direction that corresponds to the chain direction. The temperature dependence of the associated relaxation time for this series of compounds systematically exhibits a crossover between two Arrhenius laws corresponding to infinite-chain and finite-chain regimes for the SCM behavior. These isostructural hetero-spin SCMs offer a unique series of alternating [-Mn-NC-M-CN-] chains, enabling physicists to test theoretical SCM models between the Ising and Heisenberg limits. PMID:22344962

Miyasaka, Hitoshi; Madanbashi, Tomokura; Saitoh, Ayumi; Motokawa, Natsuko; Ishikawa, Ryuta; Yamashita, Masahiro; Bahr, Stefan; Wernsdorfer, Wolfgang; Clérac, Rodolphe



Assessing ATP binding and hydrolysis by NLR proteins  

PubMed Central

Summary Nucleotide-binding and leucine rich repeat domain-containing proteins (NLR) are central to the formation of many inflammasome complexes. Several inflammasome forming NLR proteins are known to be ATPases, but the nucleotide binding specificity of many remains to be characterized. The oligomerization of NLR proteins and assembly of inflammasomes require the ATP (or other nucleotide) binding activity of the NLR proteins. Quantitative and qualitative studies of the nucleotide binding properties of these proteins are useful tools in studying the regulation of inflammasome activity, and will be outlined in this Chapter.

Mo, Jinyao; Duncan, Joseph A.



Nucleotide-evoked relaxation of rat vas deferens—a possible role for endogenous ATP released upon ? 1-adrenoceptor stimulation  

Microsoft Academic Search

The possibility was tested that endogenous ATP released upon ?1-adrenoceptor activation causes relaxation of the rat vas deferens smooth muscle. ATP, 2-methylthio ATP and adenosine relaxed the vas deferens precontracted with 80 mM K+. The metabolically stable P2 receptor agonists ?,?-methylene ATP (?,?-MeATP) and adenosine 5?-O-(2-thiodiphosphate) (ADP?S) had little or no effect. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline did not

Ralph Bültmann; Klaus Starke



50 CFR 451.01 - Definitions.  

Code of Federal Regulations, 2013 CFR




50 CFR 453.02 - Definitions.  

Code of Federal Regulations, 2013 CFR




Molecular mechanism for ATP-dependent closure of the K+ channel Kir6.2.  


In the ATP-dependent K+ (KATP) channel pore-forming protein Kir6.2, mutation of three positively charged residues, R50, K185 and R201, impairs the ability of ATP to close the channel. The mutations do not change the channel open probability (Po) in the absence of ATP, supporting the involvement of these residues in ATP binding. We recently proposed that at least two of these positively charged residues, K185 and R201, interact with ATP phosphate groups to cause channel closure: the beta phosphate group of ATP interacts with K185 to initiate closure, while the alpha phosphate interacts with R201 to stabilize the channel's closed state. In the present study we replaced these three positive residues with residues of different charge, size and hydropathy. For K185 and R201, we found that charge, more than any other property, controls the interaction of ATP with Kir6.2. At these positions, replacement with another positive residue had minor effects on ATP sensitivity. In contrast, replacement of K185 with a negative residue (K185D/E) decreased ATP sensitivity much more than neutral substitutions, suggesting that an electrostatic interaction between the beta phosphate group of ATP and K185 destabilizes the open state of the channel. At R201, replacement with a negative charge (R201E) had multiple effects, decreasing ATP sensitivity and preventing full channel closure at high concentrations. In contrast, the R50E mutation had a modest effect on ATP sensitivity, and only residues such as proline and glycine that affect protein structure caused major decreases in ATP sensitivity at the R50 position. Based on these results and the recently published structure of Kir3.1 cytoplasmic domain, we propose a scheme where binding of the beta phosphate group of ATP to K185 induces a motion of the surrounding region, which destabilizes the open state, favouring closure of the M2 gate. Binding of the alpha phosphate group of ATP to R201 then stabilizes the closed state. R50 on the N-terminus controls ATP binding by facilitating the interaction of the beta phosphate group of ATP with K185 to destabilize the open state. PMID:12860923

John, Scott A; Weiss, James N; Xie, Lai-Hua; Ribalet, Bernard



The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes  

PubMed Central

Introduction Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process. Methods We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release. Results eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels. Conclusions These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.



Intracellular ATP Concentration Contributes to the Cytotoxic and Cytoprotective Effects of Adenosine  

PubMed Central

Extracellular adenosine (Ade) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway.

Guo, Haiping; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Yang, Changshan; Tang, Ping; Liu, Jinbao



Extracellular ATP in the Immune System: More Than Just a "Danger Signal"  

NSDL National Science Digital Library

Extracellular adenosine 5′-triphosphate (eATP) is ubiquitously used for cell-to-cell communication. The low concentration of eATP ([eATP]) that exists in a “halo” surrounding resting cells signals the presence of neighboring living cells. Transient increases in [eATP] are used for basic physiological signaling, namely, in the nervous and vascular systems. Larger increases in [eATP] that are associated with cell death serve as a key “danger” signal in inflammatory processes. Two studies now point to roles for ATP in the immune system: providing a costimulatory signal to T cells and driving the differentiation of intestinal T helper 17 (TH17) cells.

Alain Trautmann (France;Université Âaris Descartes REV)



Nonsynaptic and nonvesicular ATP release from neurons and relevance to neuron-glia signaling  

PubMed Central

Studies on the release of ATP from neurons began with the earliest investigations of quantal neurotransmitter release in the 1950s, but in contrast to ATP release from other cells, studies of ATP release from neurons have been narrowly constrained to one mechanism, vesicular release. This is a consequence of the prominence of synaptic transmission in neuronal communication, but nonvesicular mechanisms for ATP release from neurons are likely to have a broader range of functions than synaptic release. Investigations of activity-dependent communication between axons and myelinating glia have stimulated a search for mechanisms that could release ATP from axons and other nonsynaptic regions in response to action potential firing. This has identified volume-activated anion channels as an important mechanism in activity-dependent ATP release from axons, and renewed interest in micromechanical changes in axons that accompany action potential firing.

Fields, R. Douglas



Structure-guided simulations illuminate the mechanism of ATP transport through VDAC1.  


The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded ?-barrel with an ?-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates. PMID:24908397

Choudhary, Om P; Paz, Aviv; Adelman, Joshua L; Colletier, Jacques-Philippe; Abramson, Jeff; Grabe, Michael



Application of ATP measurements to the microbiological evaluation of a petroleum reservoir  

SciTech Connect

The objective of the work reported in this document was to determine whether the bioluminescent luciferin/luciferase based adenosine triphosphate (ATP) assay could be used as a rapid field tests for determining the presence and numbers of microorganisms in oil field fluids. The ATP-photometric technique employed is based on the ATP-mediated bioluminescent oxidation of firefly luciferin. Light production is stoichiometrically related to ATP concentration; ATP concentration is related to numbers of living organisms present in a sample. Samples used in this study comprised reservoir fluids collected from several Southern California oilfields. Based on experimental evidence, it was concluded that the ATP assay could be profitably applied to Microbially Enhanced Oil Recovery (MEOR) process monitoring and control. The theoretical basis for the assay, field-usage methodologies, and fundamentals of data interpretation are presented to make the document usable as a field manual.

Jones, P.M.



Using the Bayley-III to assess neurodevelopmental delay: which cut-off should be used?  


Background:As the latest edition of the Bayley Scales (Bayley-III) produces higher scores than its predecessor (BSID-II), there is uncertainty about how to classify moderate-severe neurodevelopmental delay. We have investigated agreement between classifications of delay made using the BSID-II and Bayley-III.Methods:BSID-II Mental Development Index (MDI) and Bayley-III cognitive and language scales were administered in 185 extremely preterm (<27?wk) children. A combined Bayley-III score (CB-III) was computed. Agreement between delay classified using MDI scores <70 and various Bayley-III cut-offs was assessed.Results:Bayley-III cognitive and language scores were close to the normative mean and were higher than BSID-II MDI scores. Nineteen (10.2%) children had MDI <70. Bayley-III scores <70 significantly underestimated the proportion with MDI <70. Bayley-III cognitive and language scores <85 had 99% agreement with MDI <70 and underestimated delay by 1.1%. CB-III scores <80 had 98% agreement and produced the same proportion with delay.Conclusion:Bayley-III cognitive and language scores <85 or CB-III scores <80 provide the best definition of moderate-severe neurodevelopmental delay for equivalence with MDI <70. CB-III scores have the advantage of producing a single continuous outcome measure but require further validation. The relative accuracy of both tests for predicting long-term outcomes requires investigation. PMID:24492622

Johnson, Samantha; Moore, Tamanna; Marlow, Neil



Purification of ATP synthase from beef heart mitochondria (F0F1) and co-reconstitution with monomeric bacteriorhodopsin into liposomes capable of light-driven ATP synthesis.  


ATP synthase was isolated from beef heart mitochondria by extraction with N,N-bis-(3-D-gluconamidopropyl)deoxycholamide or by traditional cholate extraction. The enzyme was purified subsequently by ion-exchange and gel-permeation chromatographies in the presence of glycerol and the protease inhibitor diisopropylfluorophosphate. The ATP synthase consisted of 12-14 subunits and contained three tightly bound nucleotides. The co-reconstitution of crude or purified ATP synthase with monomeric bacteriorhodopsin by the method of detergent incubation of liposomes yielded proteoliposomes capable of light-driven ATP synthesis, as detected with a luciferase system for at least 30 min. The reaction was suppressed by the inhibitors oligomycin (> 90%) and dicyclohexylcarbodiimide (85%) and by the uncoupler carbonylcyanide-p-trifluormethoxyphenylhydrazone (> 95%). The purified ATP synthase was apparently free of cytochrome impurities and of adenylate kinase activity, i.e. the enzyme exhibited light-driven ATP synthesis without the dark reaction. For the first time, this is demonstrated with purified ATP synthase from beef heart mitochondria. PMID:8269926

Deisinger, B; Nawroth, T; Zwicker, K; Matuschka, S; John, G; Zimmer, G; Freisleben, H J



Is ATP synthesized by a vacuolar-ATPase in the extremely halophilic bacteria?  

Microsoft Academic Search

The proton-dependent synthesis of ATP was demonstrated in representative members of the generaHalobacterium, Haloarcula, andHaloferax. In all cases, synthesis was not inhibited by nitrate or N-ethylmaleimide, inhibitors of the vacuolar-like ATPase found in Archaea, but was affected by azide, an inhibitor of F0F1-ATP syntheses. These observations extend the earlier observations withHalobacterium saccharovorum and suggest that ATP synthesis in these organisms

L. I. Hochstein; D. Lawson



ATP Dependence of Na+-Driven Cl-HCO3 Exchange in Squid Axons  

PubMed Central

Squid giant axons recover from acid loads by activating a Na+-driven Cl–HCO3 exchanger. We internally dialyzed axons to an intracellular pH (pHi) of 6.7, halted dialysis and monitored the pHi recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (JH) from the rate of pHi increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP]i, finding that Michaelis-Menten kinetics describes JH vs. [ATP]i, with an apparent Vmax of 15.6 pmole cm?2 s?1 and Km of 124 µM. In series 2, we examined ATP?S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP?S and GDP?S to determine if any, by themselves, could support transport. Only ATP?S (8 mM) supported acid extrusion; ATP?S also supported the HCO3?-dependent 36Cl efflux expected of a Na+-driven Cl–HCO3 exchanger. Finally, in series 3, we asked whether any nucleotide could alter JH in the presence of a background [ATP]i of ~230 µM (control JH = 11.7 pmol cm?2 s?1). We found JH was decreased modestly by 8 mM AMP-PNP (JH = 8.0 pmol cm?2 s?1) but increased modestly by 1 mM ADP?S (JH = 16.0 pmol cm?2 s?1). We suggest that ATP?S leads to stable phosphorylation of the transporter or an essential activator.

Davis, Bruce A.; Hogan, Emilia M.; Russell, John M.; Boron, Walter F.



Bioluminescent assay of microbial ATP in postmortem tissues for the estimation of postmortem interval  

Microsoft Academic Search

Summary  To study the relationship between changes of microbial ATP in four kinds of murine tissues and the postmortem interval (PMI),\\u000a healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals.\\u000a The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentration\\u000a of microbial ATP in

Qian Liu; Qing Sun; Yan Liu; Lan Zhou; Na Zheng; Liang Liu



Modulation of K channels in dialyzed squid axons. ATP-mediated phosphorylation  

PubMed Central

In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage- clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions.



Measurement and interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments  

Microsoft Academic Search

There is a widespread need for cultivation-free methods to quantify viability of natural microbial communities in aquatic environments. Adenosine tri-phosphate (ATP) is the energy currency of all living cells, and therefore a useful indicator of viability. A luminescence-based ATP kit\\/protocol was optimised in order to detect ATP concentrations as low as 0.0001nM with a standard deviation of <5%. Using this

Frederik Hammes; Felix Goldschmidt; Marius Vital; Yingying Wang; Thomas Egli



Potentiation by cadmium ion of ATP-evoked dopamine release in rat phaeochromocytoma cells.  

PubMed Central

1. The effects of cadmium ion (Cd2+) on release of dopamine and on an inward current evoked by extracellular ATP were investigated in rat phaeochromocytoma PC12 cells. 2. Cd2+ (100 microM-3 mM) potentiated the dopamine release evoked by 30 microM ATP from the cells. Cd2+ (100 microM) shifted the concentration-response curve of ATP-evoked dopamine release to the left without affecting the maximal response. 3. Suramin (30 microM) completely abolished the dopamine release evoked by 30 microM ATP but only partially inhibited the release evoked by 100 microM ATP consistent with its role as a competitive antagonist. The response evoked by 30 microM ATP in the presence of Cd2+ (300 microM) was comparable to that observed with 100 microM ATP alone; however, only the former was almost completely inhibited by suramin. 4. Cd2+ (100 microM) potentiated an inward current activated by 30 microM ATP alone. A higher concentration of Cd2+ (300 microM) had a smaller effect on amplitude potentiation but significantly prolonged the duration of the current. 5. The time-course of the ATP-evoked dopamine release was investigated using a real-time monitoring system for dopamine release. Although Cd2+ (300 microM) had little effect on the time-course of activation the ATP-evoked dopamine release, it produced a long-lasting dopamine release which slowly returned to the baseline. 6. Taken together, these observations suggest that Cd2+ enhances ATP-evoked dopamine release by affecting P2-purinoceptor/channels. The enhancement may be attributed to a Cd(2+)-dependent increase in sensitivity to ATP.

Ikeda, M.; Koizumi, S.; Nakazawa, K.; Inoue, K.; Ito, K.; Inoue, K.



Differential patterns of peroxynitrite mediated apoptosis in proximal tubular epithelial cells following ATP depletion recovery  

Microsoft Academic Search

Ischemia-reperfusion injury (IRI) is characterized by ATP depletion in the ischemic phase, followed by a rapid increase in\\u000a reactive oxygen species, including peroxynitrite in the reperfusion phase. In this study, we examined the role of peroxynitrite\\u000a on cytotoxicity and apoptosis in an in vitro model of ATP depletion-recovery. Porcine proximal tubular epithelial (LLC-PK1) cells were ATP depleted for either 2 h (2\\/2)

Vani Nilakantan; Huanling Liang; Cheryl J. Maenpaa; Christopher P. Johnson



New features of the National Cholesterol Education Program Adult Treatment Panel III lipid-lowering guidelines.  


The National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) guidelines for lipid-lowering therapy to reduce coronary heart disease (CHD) risk contain a number of features that distinguish them from the previous ATP guidelines. These new features include modifications in lipid/lipoprotein levels considered optimal, abnormal, or reflective of risk; increased focus on primary prevention through use of Framingham risk scoring to define risk in persons with multiple lipid/nonlipid risk factors; and increased focus on the association of the metabolic syndrome with CHD risk. The introduction of the category of CHD risk equivalents-including persons with atherosclerotic disease, diabetes, or 10-year CHD risk > 20% based on Framingham scoring-results in an increase over previous guidelines in the proportion of patients categorized as being at high risk and therefore eligible for more intensive low-density lipoprotein cholesterol (LDL-C)-lowering therapy. Use of the new secondary therapeutic target of non-high-density lipoprotein cholesterol should improve management of lipid risk factors in patients who have elevated triglyceride levels after LDL-C goals have been met. These new features of the NCEP ATP III guidelines should improve identification and treatment of patients with dyslipidemias associated with CHD risk. PMID:12708635

Brewer, H Bryan



ATP-Independent Hydrocarbon Formation Catalyzed by Isolated Nitrogenase Cofactors  

PubMed Central

Nitrogenase is a highly complex and uniquely versatile metalloenzyme that is capable of reducing a broad spectrum of substrates, such as dinitrogen (N2), carbon monoxide (CO) and cyanide (CN-), under ambient conditions.[1-4] The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family, both utilizing a specific reductase (Fe protein) to donate electrons to the cofactor site (FeMoco or FeVco) of a catalytic component (MoFe or VFe protein) during catalysis. The buried location of cofactor poses a challenge to electron transfer in this process, rendering it strictly dependent on ATP-assisted formation of an electron transport chain—within a complex between the reductase and the catalytic component—that extends all the way from the [Fe4S4] cluster of the former, via the P-cluster, to the cofactor site of the latter.[5] On the other hand, both FeMoco and FeVco can be extracted as intact entities into organic solvents,[6-8] spurring interest in seeking an ATP-independent reaction system, in which electrons can be directly delivered to the isolated cofactors for substrate reduction. In particular, the recent discovery that nitrogenases can reduce CO to hydrocarbons[3,4] makes it an attractive task to explore the capacity of cofactors to directly catalyze the formation of hydrocarbons from CO, as well as CN-—another carbonaceous molecule that is isoelectronic to CO.

Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W.



Catalytic subunits atp? and atp? from the Pacific white shrimp Litopenaeus vannamei F O F 1 ATP-synthase complex: cDNA sequences, phylogenies, and mRNA quantification during hypoxia  

Microsoft Academic Search

In the mitochondrial FOF1 ATP-synthase\\/ATPase complex, subunits ? and ? are part of the extrinsic portion that catalyses ATP synthesis. Since there\\u000a are no reports about genes and proteins from these subunits in crustaceans, we analyzed the cDNA sequences of both subunits\\u000a in the whiteleg shrimp Litopenaeus vannamei and their phylogenetic relationships. We also investigated the effect of hypoxia on

Oliviert Martinez-Cruz; Fernando Garcia-Carreño; Arlett Robles-Romo; Alejandro Varela-Romero; Adriana Muhlia-Almazan



Nuclear and mitochondrial subunits from the white shrimp Litopenaeus vannamei F 0 F 1 ATP-synthase complex: cDNA sequence, molecular modeling, and mRNA quantification of atp9 and atp6  

Microsoft Academic Search

We studied for the first time the ATP-synthase complex from shrimp as a model to understand the basis of crustacean bioenergetics\\u000a since they are exposed to endogenous processes as molting that demand high amount of energy. We analyzed the cDNA sequence\\u000a of two subunits of the Fo sector from mitochondrial ATP-synthase in the white shrimp Litopenaeus vannamei. The nucleus encoded

Adriana Muhlia-Almazan; Oliviert Martinez-Cruz; Fernando Garcia-Carreño; Rodrigo Arreola; Rogerio Sotelo-Mundo; Gloria Yepiz-Plascencia



[Activating effect of ATP on NO/cGMP pathway in guinea pig cochlea].  


The present investigation was to study the relationship between ATP and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. Forty healthy purebred albino guinea pigs with sensitive pryer's reflex were randomly divided into five groups. Their cochleae were dissected and perfused immediately with different solutions. For the control group, the cochleae (group 1) were perfused with artificial perilymph basal solution (APBS, containing 100 micromol/L dipyridamole, 100 micromol/L L-Arg and 1 mmol/L IBMX). Other groups were respectively perfused group 2 with 330 micromol/L ATP, group 3 with 100 micromol/L L-NNA+330 micromol/L ATP, group 4 with 10 micromol/L ODQ+330 micromol/L ATP and group 5 with 10 micromol/L A-23187. All these reagents were freshly dissolved in artificial perilymph basal solution (APBS). The cochlear tissue specimens were collected and the average cGMP content was measured with (125)I-cGMP RIA kit. The results showed that there was no significant difference in the average cochlear tissue weights among different groups. The concentration of cGMP in the cochlear tissue of the groups perfused with ATP (59.541+/-8.744 fmol/mg) and A-23187 (55.416+/-7.018 fmol/mg) was significantly higher than those of the control group (30.089+/-4.876 fmol/mg), the groups perfused with L-NNA+ATP (28.761+/-5.019 fmol/mg) and ODQ+ATP (34.209+/-13.658 fmol/mg). No significant difference was observed between the group perfused with ATP and the one with A-23187, as well as among the control group and the groups perfused respectively with L-NNA+ATP and ODQ+ATP. These results suggest that ATP elevated the concentration of cGMP in cochlear tissue while administration of nonselective nitric oxide synthase inhibitor L-NNA and soluble guanylate cyclase inhibitor ODQ could prevent the increase of cGMP concentration induced by ATP. It is indicated that ATP is involved in the activation of NO/cGMP pathway by elevating concentration in the cytoplasm of the cochlea. In turn, NO/cGMP pathway may exert a negative action on the effects of ATP. It is suggested that there is an ATP/Ca(2+)-NO/cGMP pathway in the guinea pig cochlea. ATP and NO/cGMP pathway jointly regulate the function of the cochlea. PMID:14695482

Zhao, Li-Dong; Li, Ying-Li; Li, Ning; Li, Xing-Qi



Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents.  


Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals. PMID:3913012

Odds, F C; Cheesman, S L; Abbott, A B



Periodate-oxidized ATP modulates macrophage functions during infection with Leishmania amazonensis.  


Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7(-/-) mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning. © 2014 International Society for Advancement of Cytometry. PMID:24804957

Figliuolo, V R; Chaves, S P; Santoro, G F; Coutinho, C M L M; Meyer-Fernandes, J R; Rossi-Bergmann, B; Coutinho-Silva, R



Mechanisms of ATP release and signalling in the blood vessel wall  

PubMed Central

The nucleotide adenosine 5?-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels.

Lohman, Alexander W.; Billaud, Marie; Isakson, Brant E.



Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells  

PubMed Central

The role of cytosolic ATP in exocytosis was investigated by using amperometric measurement of insulin exocytosis in pancreatic beta cells, which were stimulated with photolysis of caged Ca2+ compounds. Insulin exocytosis occurred with two rates. We found that ATP hastened and augmented the exocytosis via selective enhancement of the exocytosis with the faster rate. A nonhydrolysable analog of ATP, adenosine 5?-O-(3-thiotriphosphate), which blocks ATPase, was even more effective than ATP, indicating that the phosphorylation event occurred downstream of ATP-dependent vesicle transportation and priming. The action of ATP was eliminated by a competitive antagonist of cAMP, and by an inhibitor of adenylate cyclase. These data characterize an ATP sensing mechanism for the Ca2+-dependent exocytosis involving adenylate-cyclase, cAMP-dependent protein kinase, and, possibly, the fusion machinery itself. Thus, the fast exocytotic machinery requires both phosphorylation and Ca2+ for the final triggering and likely constitutes a distal ATP sensor for insulin exocytosis that acts in concert with ATP-sensitive K+ channels.

Takahashi, Noriko; Kadowaki, Takashi; Yazaki, Yoshio; Ellis-Davies, Graham C. R.; Miyashita, Yasushi; Kasai, Haruo



Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes.  


ATP represents a major gliotransmitter that serves as a signaling molecule for the cross talk between glial and neuronal cells. ATP has been shown to be released by astrocytes in response to a number of stimuli under nonischemic conditions. In this study, using a luciferin-luciferase assay, we found that mouse astrocytes in primary culture also exhibit massive release of ATP in response to ischemic stress mimicked by oxygen-glucose deprivation (OGD). Using a biosensor technique, the local ATP concentration at the surface of single astrocytes was found to increase to around 4 muM. The OGD-induced ATP release was inhibited by Gd(3+) and arachidonic acid but not by blockers of volume-sensitive outwardly rectifying Cl(-) channels, cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance-related protein (MRP), connexin or pannexin hemichannels, P2X(7) receptors, and exocytotic vesicular transport. In cell-attached patches on single astrocytes, OGD caused activation of maxi-anion channels that were sensitive to Gd(3+) and arachidonic acid. The channel was found to be permeable to ATP(4-) with a permeability ratio of P(ATP)/P(Cl) = 0.11. Thus, it is concluded that ischemic stress induces ATP release from astrocytes and that the maxi-anion channel may serve as a major ATP-releasing pathway under ischemic conditions. PMID:18368522

Liu, Hong-Tao; Sabirov, Ravshan Z; Okada, Yasunobu



Glycine Uptake into Barley Mesophyll Vacuoles Is Regulated but Not Energized by ATP 1  

PubMed Central

[U-14C]glycine uptake into barley (Hordeum vulgare cv Hasso) vacuoles was investigated. Glycine (2 millimolar) transport was stimulated two- to fourfold by NaATP. Stimulation was saturable with respect to ATP (1 millimolar) and linear up to 20 millimolar glycine. Stimulation by NaATP was suppressed by Mg2+ in equimolar amounts. Neither MgATP nor Mg-inorganic pyrophosphate had any effect on basal transport rate. Thus, the proton motive force can be excluded as the driving force. Uncouplers (valinomycine/carbonylcyanide-m-chlorophenylhydrazone) inhibited the basal rate up to 30% but had no influence on NaATP-stimulated uptake. Vanadate had no effect on either basal or NaATP-stimulated uptake. Nonhydrolyzable ATP analogs (adenylyl(?, ?-methylen)-diphosphate or adenylyl-imidodiphosphate) stimulated comparable to NaATP. Other nucleotides (UTP, ADP) had no effect. Some evidence exists that other amino acids (arginine, alanine, isoleucine, phenylalanine) are transported to a certain extent by a similar mechanism. The results indicate a high capacity channel-like translocator that is regulated but not energized by ATP.

Goerlach, Joern; Willms-Hoff, Indra



Identification of a new Mpl-interacting protein, Atp5d.  


Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the ? subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling. PMID:24615392

Liu, Hongyan; Zhao, Zhenhu; Zhong, Yuxu; Shan, Yajun; Sun, Xiaohong; Mao, Bingzhi; Cong, Yuwen



Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA.  

PubMed Central

The ATP concentrations allowing transcription of both heavy- and light-strand of human mtDNA in a HeLa cell mitochondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p[NH]ppA), an ATP analog with a nonhydrolyzable beta-gamma bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous ATP. Evidence was obtained indicating that p[NH]ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process. Images

Narasimhan, N; Attardi, G



Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus1  

PubMed Central

Abstract Adenosine is well known to be released during cerebral metabolic stress and is believed to be neuroprotective. ATP release under similar circumstances has been much less studied. We have now used biosensors to measure and compare in real time the release of ATP and adenosine during in vitro ischaemia in hippocampal slices. ATP release only occurred following the anoxic depolarisation, whereas adenosine release was apparent almost immediately after the onset of ischaemia. ATP release required extracellular Ca2+. By contrast adenosine release was enhanced by removal of extracellular Ca2+, whilst TTX had no effect on either ATP release or adenosine release. Blockade of ionotropic glutamate receptors substantially enhanced ATP release, but had only a modest effect on adenosine release. Carbenoxolone, an inhibitor of gap junction hemichannels, also greatly enhanced ischaemic ATP release, but had little effect on adenosine release. The ecto-ATPase inhibitor ARL 67156, whilst modestly enhancing the ATP signal detected during ischaemia, had no effect on adenosine release. Adenosine release during ischaemia was reduced by pre-treament with homosysteine thiolactone suggesting an intracellular origin. Adenosine transport inhibitors did not inhibit adenosine release, but instead they caused a twofold increase of release. Our data suggest that ATP and adenosine release during ischaemia are for the most part independent processes with distinct underlying mechanisms. These two purines will consequently confer temporally distinct influences on neuronal and glial function in the ischaemic brain.

Frenguelli, Bruno G; Wigmore, Geoffrey; Llaudet, Enrique; Dale, Nicholas



New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La  

SciTech Connect

E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.



ATP-responsive controlled release system using aptamer-functionalized mesoporous silica nanoparticles.  


Adenosine-5'-triphosphate (ATP) is a multifunctional nucleotide, which plays a vital role in many biological processes, including muscle contraction, cells functioning, synthesis and degradation of important cellular compounds, and membrane transport. Thus, the development of ATP-responsive controlled release system for bioorganism application is very significative. Here, an original and facile ATP-responsive controlled release system consisting of mesoporous silica nanoparticles (MSN) functionalized with an aptamer as cap has been designed. In this system, the ATP aptamer was first hybridized with arm single-stranded DNA1 (arm ssDNA1) and arm single-stranded DNA2 (arm ssDNA2) to form the sandwich-type DNA structure and then grafted onto the MSN surface through click chemistry approach, resulting in blockage of pores and inhibition of guest molecules release. In the presence of ATP, the ATP aptamer combined with ATP and got away from the pore, leaving the arm ssDNA1 and ssDNA2 on the surface of MSN. The guest molecules can be released because single-stranded DNA is flexible. The release of the guest molecules from this system then can be triggered by the addition of ATP. As a proof-of-principle, Ru(bipy)(3)(2+) was selected as the guest molecules, and the ATP-responsive loading and release of Ru(bipy)(3)(2+) have been investigated. The results demonstrate that the system had excellent loading efficiency (215.0 ?mol g(-1) SiO(2)) and the dye release percentage can reach 83.2% after treatment with 20 mM ATP for 7 h. Moreover, the ATP-responsive behavior shows high selectivity with ATP analogues. However, the leakage of Ru(bipy)(3)(2+) molecule is neglectable if ATP was not added, indicating an excellent capping efficiency. Interestingly, this system can respond not only to the commercial ATP but also to the ATP extracted from living cells. By the way, this system is also relatively stable in mouse serum solution at 37 °C. This proof of concept might promote the application of ATP-responsive devices and can also provide an idea to design various target-responsive systems using other aptamers as cap. PMID:22889263

He, Xiaoxiao; Zhao, Yingxiang; He, Dinggeng; Wang, Kemin; Xu, Fengzhou; Tang, Jinlu



A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase.  


Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein. PMID:23827679

Lee, Eun-Jin; Pontes, Mauricio H; Groisman, Eduardo A



Red blood cell ATP/ADP & nitric oxide: The best vasodilators in diabetic patients  

PubMed Central

Background Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose) levels that result from defects in insulin secretion, or action, or both. Inspired by previous report the release of ATP from RBCs, which may participate in vessel dilation by stimulating NO production in the endothelium through purinergic receptor signaling and so, the aim of this study is to clearly determined relationship between RBC ATP/ADP ratio with nitric oxide. Methods The ATP/ADP ratio of erythrocytes among four groups of normal individuals (young & middle age), athletes’ subjects and diabetic patients were compared and the relationship between ATP/ADP ratio and NO level of plasma was determined with AVOVA test and bioluminescence method. Results ATP/ADP level in four groups normal (young & middle age), athletes, diabetes] are measured and analyzed with ANOVA test that show a significant difference between groups (P-value < 0.001). A significant positive correlation was found between RBC ATP/ADP content (r = 0.705; P < 0.001). Plasma NO content is also analyzed with ANOVA test which shows a significant difference between groups. Conclusion In this study, a positive relationship between RBC ATP/ADP ratio and NO was found. Based on the obtained result, higher RBC ATP/ADP content may control the ratio of plasma NO in different individuals, also this results show that ATP can activate endothelial cells in NO production and is a main factor in releasing of NO from endothelial cells.



Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release.  


RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold. PMID:21690355

Forsyth, Alison M; Wan, Jiandi; Owrutsky, Philip D; Abkarian, Manouk; Stone, Howard A




Grouping Page:  FIRE III Main Grouping:  Arctic Cloud Experiment Description:  The First ... in conjunction with the Surface Heat Budget of the Arctic Ocean (SHEBA) Experiment. The FIRE-ACE focused on all aspects of Arctic cloud ...



Title III Storm Signals.  

ERIC Educational Resources Information Center

Financial problems, and even the survival, of developing institutions funded by Title III are reported. Issues include the HEW freeze on grant money and problems with student financial aid from the government. (LBH)

Holsendolph, Ernest



Tobacco industry influence on the definition of tobacco related disorders by the American Psychiatric Association  

PubMed Central

Objective: The Diagnostic and statistical manual of mental disorders, third edition (DSM-III), published by the American Psychiatric Association (APA) in 1980, included the first official definitions by the APA of tobacco dependence and tobacco withdrawal. Tobacco industry efforts to influence the DSM-III were investigated. Method: Searches of previously secret tobacco industry documents, primarily the University of California San Francisco Legacy Tobacco Documents Library and British American Tobacco collections. Additional information was collected through discussions with editors of DSM-III, and library and general internet searches. Results: The tobacco companies regarded the inclusion of tobacco dependence as a diagnosis in DSM-III as an adverse event. It worked to influence the content of the DSM-III and its impact following publication. These efforts included public statements and private lobbying of DSM-III editors and high ranking APA officers by prominent US psychiatrists with undisclosed ties to the tobacco industry. Following publication of DSM-III, tobacco companies contracted with two US professors of psychiatry to organise a conference and publish a monograph detailing controversies surrounding DSM-III. Conclusions: The tobacco industry and its allies lobbied to narrow the definition of tobacco dependence in serial revisions of DSM-III. Following publication of DSM-III, the industry took steps to try to mitigate its impact. These actions mirror industry tactics to influence medical research and policy in various contexts worldwide. Such tactics slow the spread of a professional and public understanding of smoking and health that otherwise would reduce smoking, smoking induced disease, and tobacco company profits.

Neuman, M; Bitton, A; Glantz, S



34 CFR 101.131 - Definitions.  

Code of Federal Regulations, 2013 CFR

...2013-07-01 2013-07-01 false Definitions. 101.131 Section 101.131 Education Regulations of the Offices of the Department...UNDER PART 100 OF THIS TITLE Definitions § 101.131 Definitions. The definitions contained...



Calcium handling and purinoceptor subtypes involved in ATP-induced contraction in rat small mesenteric arteries.  

PubMed Central

1. The relationship between the stimulation of ATP receptors, the increase in intracellular free calcium concentration ([Ca2+]i; measured using the fluorescent indicator fura-2), contraction and the subtypes of purinoceptors involved were investigated in the small mesenteric artery of the rat. 2. In normal physiological solution, ATP (0.001-3 mM) caused concentration-dependent increases in both [Ca2+]i and contraction. Both responses produced by ATP (1 mM) were inhibited by 50% in the presence of nitrendipine (1 microM) and were abolished in the presence of nitrendipine plus SK&F 96365 (30 microM). 3. In Ca(2+)-free medium, ATP (3 mM) elicited a transient increase in both [Ca2+]i and tension which were abolished by caffeine and decreased by 65% by thapsigargin (1 microM). Moreover, ATP (1 and 3 mM) produced increases in the [3H]D-myo-inositol 1,4,5-trisphosphate ([3H]IP3) content of vessels in a concentration-dependent manner. 4. Treatment of the vessels with Bordetella pertussis toxin (PTX) inhibited contractions to ATP linked to the influx of calcium through nitrendipine-sensitive mechanisms, but not those linked to the release of Ca2+ from intracellular stores nor the capacity of ATP in increasing IP3 content of the vessels. 5. The order of potency of ATP and its analogues in eliciting contraction was alpha, beta-methylene-ATP (alpha, beta-MeATP) > 2-methylthio-ATP (2-MeSATP) > ATP = ADP. The response to ATP was inhibited by suramin. Reactive Blue 2 (up to 100 microM) did not affect the contractile response to ATP. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) and alpha, beta-MeATP abolished the response to low concentrations of ATP and reduced contractions elicited by high concentrations of ATP. 6. After blockade of P2X-purinoceptors with PPADS, the order of potency of ATP and its analogues was 2-MeSATP > ATP = ADP. UTP produced concentration-dependent contractions which were not affected by suramin, Reactive Blue 2, PPADS or alpha, beta-MeATP, suggesting the presence of P2U-purinoceptors. 7. The results suggest that low concentrations of ATP activate P2X-purinoceptors and produce an influx of calcium through both voltage-dependent calcium channels sensitive to nitrendipine and through receptor-operated calcium channels sensitive to SK&F 96365. High concentrations of ATP activate P2Y-purinoceptors which promote firstly a nitrendipine-sensitive calcium influx via a PTX-sensitive G protein and secondly a release of Ca2+ from an internal source via the production of IP3.

Lagaud, G J; Stoclet, J C; Andriantsitohaina, R



ATP modulates anti-IgE-induced release of histamine from human lung mast cells.  


Adenosine 5'-triphosphate (ATP) is released from the cytoplasm under physiologic and pathophysiologic conditions and enters the extracellular space, where it acts on a group of recently cloned cell-surface receptors termed P2-purinoceptors (subtypes P2X and P2Y). We examined the effects of extracellular ATP, uridine triphosphate (UTP), the stable ATP analogues alpha,betamethylene-ATP (alpha,betamATP), beta,gammamethylene-ATP (beta,gammamATP), and 2-methylthio-ATP (2mSATP), and adenosine (10(-6)-10(-3) M) on histamine release from human lung mast cells (HLMC) induced by anti-IgE and the calcium ionophore A23187. None of the nucleotides or adenosine directly induced histamine release. Adenosine exhibited a bimodal effect, enhancing histamine release at 10(-6) to 10(-4) M (P > 0.05, NS) and inhibiting it at 10(-3) M (P < 0.05). ATP (10(-4) M) enhanced anti-IgE-induced histamine release (10.9 +/- 2.7% to 19. 2 +/- 2.9%, n = 20, P < 0.01), but not ionophore A23187-induced histamine release (n = 10). The adenine nucleotides consistently enhanced anti-IgE-induced histamine release; the rank order for this action was: ATP > 2mSATP > alpha,betamATP > beta,gammamATP, suggesting mediation by a P2Y-purinoceptor subtype. The selective P2X purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid failed to influence the effect of ATP, further supporting P2Y-purinoceptor mediation of anti-IgE-induced histamine release. UTP, an agonist at P2Y-purinoceptors, also significantly enhanced anti-IgE-induced histamine release. Application of the reverse transcription-polymerase chain reaction indicated that HLMC constitutively express the messenger RNAs encoding the P2Y1- and P2Y2-purinoceptor subtypes, and not that encoding the P2X7-purinoceptor (i.e., P2Z), a subtype implicated in ATP-induced histamine release in rodent peritoneal mast cells. The data produced in the study suggest that ATP plays an important modulatory role in histamine release from HLMC, and that it may therefore be mechanistically involved in human allergic/asthmatic reactions. PMID:10030852

Schulman, E S; Glaum, M C; Post, T; Wang, Y; Raible, D G; Mohanty, J; Butterfield, J H; Pelleg, A



Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel  

PubMed Central

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca2+ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N3ATP-2?,3?-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC50?=?0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.

Popova, Olga B.; Baker, Mariah R.; Tran, Tina P.; Le, Tri; Serysheva, Irina I.



Mechanism of prolonged vasorelaxation to ATP in the rat isolated mesenteric arterial bed  

PubMed Central

This study investigated the mechanism of prolonged relaxation to ATP in the rat isolated perfused mesenteric arterial bed. In methoxamine pre-constricted preparations, ATP elicited dose-dependent, endothelium-dependent, rapid relaxation at 5?pmol?–?0.05??mol (Rmax 76±5.6%, pD2 9.2±0.2), and contraction, followed by prolonged endothelium-independent vasorelaxation at 0.05, 0.5 and 5??mol (56±3.0, 87±2.9 and 85±4.6%). Suramin (100??M), attenuated rapid (pD2 7.8±0.1) and prolonged relaxation to ATP. The selective P2 receptor antagonist PPADS (10??M) reduced prolonged, but not rapid relaxation. Neither phase of relaxation was affected by 8-sulphophenyltheophylline (1??M) or indomethacin (10??M). ?,?-methylene ATP (?,?-meATP; 10??M) attenuated prolonged relaxation to ATP (relaxations at 0.05 and 0.5??mol were 25±8.3 and 48±9.0%, respectively). ?,?-meATP blocked contractions and revealed rapid relaxation to ATP at 0.05?–?5??mol. Capsaicin pre-treatment did not affect either phase of vasorelaxation to ATP. ?,?-meATP (10??M) had no effect on vasorelaxation mediated by electrical stimulation of capsaicin-sensitive sensory nerves. High K+ (25?mM) attenuated prolonged relaxation to ATP (21±2.6 and 64±5.8%, at 0.05 and 0.5??mol, respectively), but had no effect on rapid relaxation. Ouabain (1?mM), an inhibitor of Na+/K+-ATPase, and glibenclamide (10??M), an inhibitor of KATP channels, also attenuated prolonged relaxation to ATP. Charybdotoxin (100?nM), a selective inhibitor of KCa channels, and tetraethylammonium (10?mM) had no effect on rapid or prolonged relaxations. These results show that the prolonged phase of vasorelaxation to ATP in the rat isolated mesenteric arterial bed, which may be mediated by P2Y receptors, is endothelium-independent, involves activation of Na+/K+-ATPase and KATP channels, and is inhibited by ?,?-meATP. Neither prolonged nor rapid vasorelaxation to ATP involves capsaicin-sensitive sensory nerves, adenosine P1 receptors, prostanoids or KCa channels.

Ralevic, Vera



ATP secretion in the male reproductive tract: essential role of CFTR.  


Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations. PMID:22711960

Ruan, Ye Chun; Shum, Winnie W C; Belleannée, Clémence; Da Silva, Nicolas; Breton, Sylvie



Macula densa basolateral ATP release is regulated by luminal [NaCl] and dietary salt intake.  


One component of the macula densa (MD) tubuloglomerular feedback (TGF) signaling pathway may involve basolateral release of ATP through a maxi-anion channel. Release of ATP has previously been studied during a maximal luminal NaCl concentration ([NaCl](L)) stimulus (20-150 mmol/l). Whether MD ATP release occurs during changes in [NaCl](L) within the physiological range (20-60 mmol/l) has not been examined. Also, because TGF is known to be enhanced by low dietary salt intake, we examined the pattern of MD ATP release from salt-restricted rabbits. Fluorescence microscopy, with fura 2-loaded cultured mouse mesangial cells as biosensors, was used to assess ATP release from the isolated, perfused thick ascending limb containing the MD segment. The mesangial biosensor cells, which contain purinergic receptors and elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) on ATP binding, were placed adjacent to the MD basolateral membrane. Elevations in [NaCl](L) between 0 and 80 mmol/l, in 20-mmol/l increments, caused stepwise increases in [Ca(2+)](i), with the highest increase at [NaCl](L) of approximately 60 mmol/l. Luminal furosemide at 10(-4) mol/l blocked ATP release, which suggests that the efflux of ATP required MD Na-2Cl-K cotransport. A low-salt diet for 1 wk increased the magnitude of [NaCl](L)-dependent elevations in biosensor [Ca(2+)](i) by twofold, whereas high-salt intake had no effect. In summary, ATP release occurs over the same range of [NaCl](L) (20-60 mmol/l) previously reported for TGF responses, and, similar to TGF, ATP release was enhanced by dietary salt restriction. Thus these two findings are consistent with the role of MD ATP release as a signaling component of the TGF pathway. PMID:14749255

Komlosi, Peter; Peti-Peterdi, Janos; Fuson, Amanda L; Fintha, Attila; Rosivall, Laszlo; Bell, Phillip Darwin