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Sample records for brain atp synthesis

  1. Multimodal neuroimaging provides a highly consistent picture of energy metabolism, validating 31P MRS for measuring brain ATP synthesis

    International Nuclear Information System (INIS)

    Neuroimaging methods have considerably developed over the last decades and offer various noninvasive approaches for measuring cerebral metabolic fluxes connected to energy metabolism, including PET and magnetic resonance spectroscopy (MRS). Among these methods, 31P MRS has the particularity and advantage to directly measure cerebral ATP synthesis without injection of labeled precursor. However, this approach is methodologically challenging, and further validation studies are required to establish 31P MRS as a robust method to measure brain energy synthesis. In the present study, we performed a multimodal imaging study based on the combination of 3 neuroimaging techniques, which allowed us to obtain an integrated picture of brain energy metabolism and, at the same time, to validate the saturation transfer 31P MRS method as a quantitative measurement of brain ATP synthesis. A total of 29 imaging sessions were conducted to measure glucose consumption (CMRglc), TCA cycle flux (VTCA), and the rate of ATP synthesis (VATP) in primate monkeys by using 18F-FDG PET scan, indirect 13C MRS, and saturation transfer 31P MRS, respectively. These 3 complementary measurements were performed within the exact same area of the brain under identical physiological conditions, leading to: CMRglc = 0.27 ± 0.07 μmol.g-1.min-1, VTCA = 0.63 ± 0.12 μmol.g-1.min-1, and VATP = 7.8 ± 2.3 μmol.g-1.min-1. The consistency of these 3 fluxes with literature and, more interestingly, one with each other, demonstrates the robustness of saturation transfer 31P MRS for directly evaluating ATP synthesis in the living brain. (authors)

  2. Thermodynamics of proton transport coupled ATP synthesis.

    Science.gov (United States)

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°ref=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pHin or pHout) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F0 to the catalytic nucleotide binding sites on the β-subunits in F1, is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. PMID:26940516

  3. ATP Synthesis in the Extremely Halophilic Bacteria

    Science.gov (United States)

    Hochstein, Lawrence I.; Morrison, David (Technical Monitor)

    1994-01-01

    The proton-translocating ATPases are multimeric enzymes that carry out a multitude of essential functions. Their origin and evolution represent a seminal event in the early evolution of life. Amino acid sequences of the two largest subunits from archaeal ATPases (A-ATPases), vacuolar ATPases (V-ATPases), and FOF1-ATP syntheses (FATPases) suggest these ATPases evolved from an ancestral vacuolar-like ATP syntheses. A necessary consequence of this notion is that the A-ATPases are ATP syntheses. With the possible exception of the A-ATPase from Halobacterium salinarium. no A-ATPase has been demonstrated to synthesize ATP. The evidence for this case is dubious since ATP synthesis occurs only when conditions are distinctively unphysiological. We demonstrated that ATP synthesis in H.saccharovorum is inconsistent with the operation of an A-type ATPase. In order to determine if this phenomenon was unique to H. saccharovorum, ATP synthesis was examined in various extremely halophilic bacteria with the goal of ascertaining if it resembled what occurred in a. saccharovorum, or was consistent with the operation of an A-type ATPase. A-, V-, and F-type ATPases respond singularly to certain inhibitors. Therefore, the effect of these inhibitors on ATP synthesis in several extreme halophiles was determined. Inhibitors that either blocked or collapsed proton-gradients inhibited the steady state synthesis of ATP thus verifying that synthesis took place at the expense of a proton gradient. Azide, an inhibitor of F-ATPases inhibited ATP synthesis. Since the arginine-dependent synthesis of ATP, which occurs by way of substrate-level phosphorylation, was unaffected by azide, it was unlikely that azide acted as an "uncoupler." N -ethylmaleimide and nitrate, which inhibit V- and A-ATPases, either did not inhibit ATP synthesis or resulted in higher steady-state levels of ATP. These results suggest there are two types of proton-motive ATPases in the extreme halophiles (and presumably in other

  4. Magnetic field affects enzymatic ATP synthesis.

    Science.gov (United States)

    Buchachenko, Anatoly L; Kuznetsov, Dmitry A

    2008-10-01

    The rate of ATP synthesis by creatine kinase extracted from V. xanthia venom was shown to depend on the magnetic field. The yield of ATP produced by enzymes with 24Mg2+ and 26Mg2+ ions in catalytic sites increases by 7-8% at 55 mT and then decreases at 80 mT. For enzyme with 25Mg2+ ion in a catalytic site, the ATP yield increases by 50% and 70% in the fields 55 and 80 mT, respectively. In the Earth field the rate of ATP synthesis by enzyme, in which Mg2+ ion has magnetic nucleus 25Mg, is 2.5 times higher than that by enzymes, in which Mg2+ ion has nonmagnetic, spinless nuclei 24Mg or 26Mg. Both magnetic field effect and magnetic isotope effect demonstrate that the ATP synthesis is an ion-radical process, affected by Zeeman interaction and hyperfine coupling in the intermediate ion-radical pair. PMID:18774801

  5. Compartmentalized ATP synthesis in skeletal muscle triads.

    Science.gov (United States)

    Han, J W; Thieleczek, R; Varsányi, M; Heilmeyer, L M

    1992-01-21

    Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads. PMID:1731894

  6. Synthesis and fluorescence characteristics of ATP-based FRET probes

    OpenAIRE

    Hardt, Normann; Hacker, Stephan M.; Marx, Andreas

    2013-01-01

    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 γ-position is a very promising starting point for the design of these probes. We synt...

  7. Pyrazinoic Acid Decreases the Proton Motive Force, Respiratory ATP Synthesis Activity, and Cellular ATP Levels▿†

    OpenAIRE

    Lu, P.; Haagsma, A.C.; Pham, H.; Maaskant, J. J.; Mol, S; Lill, H.; Bald, D

    2011-01-01

    Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

  8. Synthesis and fluorescence characteristics of ATP-based FRET probes.

    Science.gov (United States)

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

    2013-12-28

    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 γ-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 γ- 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

  9. ATP synthesis during exogenous NADH oxidation. A reappraisal.

    Science.gov (United States)

    Bernardi, P; Azzone, G F

    1982-01-20

    This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c. PMID:6275889

  10. ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    obese and eight subjects with type 2 diabetes precultured under normophysiological conditions. Furthermore, mitochondria were isolated and ATP production was measured by luminescence at baseline and during acute insulin stimulation with or without concomitant ATP utilization by hexokinase. Mitochondrial...... mass and the ATP synthesis rate, neither at baseline nor during acute insulin stimulation, were not different between groups. The ratio of ATP synthesis rate at hexokinase versus ATP synthesis rate at baseline was lower in diabetic mitochondria compared to lean mitochondria. Thus the lower content...

  11. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  12. The two-hit hypothesis for neuroinflammation: role of exogenous ATP in modulating inflammation in the brain

    Directory of Open Access Journals (Sweden)

    Bernd L. Fiebich

    2014-09-01

    Full Text Available Brain inflammation is a common occurrence following responses to varied insults such as bacterial infections, stroke, traumatic brain injury and neurodegenerative disorders. A common mediator for these varied inflammatory responses is prostaglandin E2 (PGE2, produced by the enzymatic activity of cyclooxygenases (COX 1 and 2. Previous attempts to reduce neuronal inflammation through COX inhibition, by use of nonsteroidal anti-inflammatory drugs (NSAIDs, have met with limited success. We are proposing the two-hit model for neuronal injury – an initial localized inflammation mediated by PGE2 (first hit and the simultaneous release of adenosine triphosphate (ATP by injured cells (second hit, which significantly enhances the inflammatory response through increased synthesis of PGE2. Several evidences on the role of exogenous ATP in inflammation have been reported, including contrary instances where extracellular ATP reduces inflammatory events. In this review, we will examine the current literature on the role of P2 receptors, to which ATP binds, in modulating inflammatory reactions during neurodegeneration. Targeting the P2 receptors, therefore, provides a therapeutic alternative to reduce inflammation in the brain. P2 receptor-based anti-inflammatory drugs (PBAIDs will retain the activities of essential COX enzymes, yet will significantly reduce neuroinflammation by decreasing the enhanced production of PGE2 by extracellular ATP.

  13. An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts

    DEFF Research Database (Denmark)

    Pateraki, Irini; Renato, Marta; Azcõn-Bieto, Joaquín;

    2013-01-01

    synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in...... non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report...... the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to...

  14. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    Directory of Open Access Journals (Sweden)

    Ya Hui Hung

    2013-08-01

    Full Text Available Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-Type ATPases (copper-ATPases, ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  15. Mechanical Stress and ATP Synthesis are coupled by Mitochondrial Oxidants in Articular Cartilage

    OpenAIRE

    Wolff, Katherine J; Ramakrishnan, Prem S.; Brouillette, Marc J.; Journot, Brice; Mckinley, Todd O; Buckwalter, JA; Martin, James A.

    2012-01-01

    Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for...

  16. Coassembly of Photosystem II and ATPase as Artificial Chloroplast for Light-Driven ATP Synthesis.

    Science.gov (United States)

    Feng, Xiyun; Jia, Yi; Cai, Peng; Fei, Jinbo; Li, Junbai

    2016-01-26

    Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-to-ATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control. PMID:26615669

  17. The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2011-01-01

    or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological...... conditions in order to verify intrinsic impairments. To resemble dynamic equilibrium present in whole cells between ATP synthesis and utilization, ATP was measured in the presence of an ATP consuming enzyme, hexokinase, under steady state. Mitochondria were isolated using an affinity based method which...... selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects...

  18. Blood-Brain Barrier Active Efflux Transporters: ATP-Binding Cassette Gene Family

    OpenAIRE

    Löscher, Wolfgang; Potschka, Heidrun

    2005-01-01

    Summary: The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from potentially harmful endogenous and exogenous substances. BBB active drug efflux transporters of the ATP-binding cassette (ABC) gene family are increasingly recognized as important determinants of drug distribution to, and elimination from, the CNS. The ABC efflux transporter P-glycoprotein (Pgp) has been demonstrated as a key element of the BBB that can actively transport a huge variety of lip...

  19. ATP P2x receptors studied by quantitative autoradiography of [3H]α,β- methylene-ATP binding in rat brain

    International Nuclear Information System (INIS)

    Full text: P2x receptors are ligand-gated cationic channels widely distributed in the central and peripheral nervous systems. The probable natural P2x ligand is ATP but the 3H-labelled form of the stable ATP analogue α,β-methylene-ATP is the only compound currently used in radioligand binding studies of P2x receptors. In order to further test its specificity for P2x receptor binding sites in the CNS we have examined the effects of several ATP analogues, and other ATP-related substances on the binding of [3]α,β-methylene-ATP to fresh-frozen sections of rat brain. Sprague-Dawley rats were decapitated under halothane anaesthesia (5% in the mixture of N2C/O2, 65:35), 20 μM thick brain sections were incubated in the presence of 10 μM [3H]α,β-methylene-ATP and 2.5 μM Ca2+ -in 50 mM Tris-HCl buffer. Autoradiograms were evaluated by quantitative densitometry. [3H]α,β-Methylene-ATP binding was sensitive to the P2 antagonist suramin (IC50 ∼ 20 μM) but it was only moderately inhibited by Reactive Blue 2 and related dyes (IC50 - 200 - 400 μM ). Two ATP analogues (3'-O-(trinitrophenyl)-adenosine-5'-triphosphate and β,γ-imido-ATP) produced IC50 -1-2 μM but β,γ-methylene-ATP was less potent. ATP analogues with other than adenine residues (inosine-5'-triphosphate, guanosine-5'-triphosphate, uridine-5'-triphosphate and cytidine-5'-triphosphate) were inactive. Cations (K+, Rb+, Cs+ and Mg2+ at 5 mM and Na+ at 150 mM) moderately reduced [3H]α,β-methylene-ATP binding but HgCl2 and p-chloromercuriphenyl sulphonate caused strong inhibitions. Several compounds known to interact with other ATP binding sites (ATPases: ouabain, thapsigargin; ATP or adenosine receptors: adenosine, 2-Cl-ATP, 2-methyl-S-ATP) and cationic channels (glibenclamide, dantrolene) had no effect. We conclude that [3H]α,β-methylene-ATP at low μM concentrations binds predominantly to P2x receptors. Copyright (1998) Australian Neuroscience Society

  20. Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange

    International Nuclear Information System (INIS)

    The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO2 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form

  1. Optimization time synthesis of nucleotide labelled [γ-32P]-ATP

    International Nuclear Information System (INIS)

    Adenosine triphosphate-labelled with γ-32P([γ-32p]-ATP) has been widely used in the biotechnology research, usually as a tracer to study aspects of physiological and pathological processes. In order to support biotechnology research in Indonesia, a process for production of [γ-32P]-ATP with enzymatic reaction was used as precursors DL-glyceraldehydde 3-phosphate, Adenosine Diphosphate (ADP) and H332PO4, and enzyme glyceraldehid 3-phosphate dehydrogenase, 3-phosphoglyceryc phosphokinase and lactate dehydrogenase. Optimization of incubation time labeled nucleotide synthesis process is performed to find the optimum conditions, in terms of the most advantageous time in the synthesis process. With the success of the synthesis and optimization is done incubation time of synthesis labeled nucleotide, the result suggested can be used for producing [γ-32P] -ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia. (author)

  2. Mechanical stress and ATP synthesis are coupled by mitochondrial oxidants in articular cartilage.

    Science.gov (United States)

    Wolff, Katherine J; Ramakrishnan, Prem S; Brouillette, Marc J; Journot, Brice J; McKinley, Todd O; Buckwalter, Joseph A; Martin, James A

    2013-02-01

    Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5 Hz with amplitude of 0.25 MPa for 1 h. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase-based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation. PMID:22930474

  3. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    Science.gov (United States)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  4. ATP increases the migration of microglia across the brain endothelial cell monolayer.

    Science.gov (United States)

    Maeda, Tomoji; Inagaki, Manato; Fujita, Yu; Kimoto, Takehiro; Tanabe-Fujimura, Chiaki; Zou, Kun; Liu, Junjun; Liu, Shuyu; Komano, Hiroto

    2016-04-01

    The cerebral microcapillary endothelium, known as the blood-brain barrier (BBB), acts as a barrier between the blood and the interstitial fluid of the brain. The BBB therefore controls the passage of nutrients into the central nervous system (CNS). Microglia show a specific affinity for migration into the CNS, and this migration appears to occur independently of BBB integrity. To study the migration of microglia across the BBB, we developed an in vitro co-culture system of mouse brain endothelial cells (MBECs) and Ra2 microglia using Transwell inserts. We first investigated the influence of microglia or ATP, a microglial chemotactic factor, on MBEC barrier integrity. The addition of microglia or ATP led to the disruption of the MBEC monolayer and significantly decreased barrier function as measured by trans-endothelial electrical resistance (TEER) and electric cell-substrate impedance sensing (ECIS). Furthermore, ATP promoted the migration of microglia but not macrophages across the MBEC monolayer. An inhibitor of matrix metalloproteinases (MMPs) decreased the transmigration of microglia in our system, indicating that MMPs play a role in microglial chemotaxis. We specifically identify a role for microglia-derived MMP-2. In conclusion, we offer evidence that microglia migration across the brain endothelial cell monolayer is increased in the presence of ATP in a manner that involves MMP secretion. PMID:26934979

  5. Inhibition of ATP synthesis by fenbufen and its conjugated metabolites in rat liver mitochondria.

    Science.gov (United States)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-03-01

    Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen-S-glutathione thioester (F-SG) were found to be more potent inhibitors compared to parent fenbufen (F), whereas fenbufen-O-carnitine (F-carn), fenbufen-glycine (F-gly) and fenbufen-N-acetyl lysine amide (F-NAL) were less potent compared to fenbufen. Fenbufen-CoA thioester (F-CoA) was equally potent as fenbufen in inhibiting ATP synthesis. Fenbufen showed time and concentration dependent inhibition of ATP synthesis with Kinact of 4.4 min(-1) and KI of 0.88 μM and Kinact/KI ratio of 5.01 min(-1) μM(-1). Data show that fenbufen did not act through opening MPT pore, nor did incubation of mitochondria with reduced GSH and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria. The results from the present experiments provide evidence that fenbufen and its metabolites could be involved in mitochondrial toxicity through inhibition of ATP synthesis. PMID:26612354

  6. Iptakalim protects against hypoxic brain injury through multiple pathways associated with ATP-sensitive potassium channels.

    Science.gov (United States)

    Zhu, H-L; Luo, W-Q; Wang, H

    2008-12-10

    The rapid and irreversible brain injury produced by anoxia when stroke occurs is well known. Cumulative evidence suggests that the activation of neuronal ATP-sensitive potassium (KATP) channels may have inherent protective effects during cerebral hypoxia, yet little information regarding the therapeutic effects of KATP channel openers is available. We hypothesized that pretreatment with a KATP channel opener might protect against brain injury induced by cerebral hypoxia. In this study, adult Wistar rats were treated with iptakalim, a new KATP channel opener, which is selective for SUR2 type KATP channels, by intragastric administration at doses of 2, 4, or 8 mg/kg/day for 7 days before being exposed to simulated high altitude equivalent to 8000 m in a decompression chamber for 8 h leading to hypoxic brain injury. By light and electron microscopic images, we observed that hypobaric hypoxia-induced brain injury could be prevented by pretreatment with iptakalim. It was also observed that the permeability of the blood-brain barrier, water content, Na+ and Ca2+ concentration, and activities of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase in rat cerebral cortex were increased and the gene expression of the occludin or aquaporin-4 was down- or upregulated respectively, which could also be prevented by the pretreatment with iptakalim at doses of 2, 4, or 8 mg/kg in a dose-dependent manner. Furthermore, we found that in an oxygen-and-glucose-deprived model in ECV304 cells and rat cortical astrocytes, pretreatment with iptakalim significantly increased survived cell rates and decreased lactate dehydrogenate release, which were significantly antagonized by glibenclamide, a K(ATP) channel blocker. We conclude that iptakalim is a promising drug that may protect against brain injury induced by acute hypobaric hypoxia through multiple pathways associated with SUR2-type K(ATP) channels, suggesting a new therapeutic strategy for stroke treatment. PMID:18951957

  7. An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis

    OpenAIRE

    Poehlein, Anja; Schmidt, Silke; Kaster, Anne-Kristin; Goenrich, Meike; Vollmers, John; Thürmer, Andrea; Bertsch, Johannes; Schuchmann, Kai; Voigt, Birgit; Hecker, Michael; Daniel, Rolf; Thauer, Rudolf K.; Gottschalk, Gerhard; Müller, Volker

    2012-01-01

    Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potenti...

  8. Stoichiometry of vectorial H+ movements coupled to electron transport and to ATP synthesis in mitochondria.

    Science.gov (United States)

    Alexandre, A; Reynafarje, B; Lehninger, A L

    1978-11-01

    In order to verify more directly our earlier measurements showing that, on the average, close to four vectorial H(+) are rejected per pair of electrons passing each of the three energy-conserving sites of the mitochondrial electron transport chain, direct tests of the H(+)/2e(-) ratio for sites 2 and 3 were carried out in the presence of permeant charge-compensating cations. Site 2 was examined by utilizing succinate as electron donor and ferricyanide as electron acceptor from mitochondrial cytochrome c; the directly measured H(+)/2e(-) ratio was close to 4. Energy-conserving site 3 was isolated for study with ferrocyanide or ascorbate plus tetramethylphenylenediamine as electron donors to cytochrome c and with oxygen as electron acceptor. The directly measured H(+)/2e(-) ratio for site 3 was close to 4. The H(+)/ATP ratio (number of vectorial H(+) ejected per ATP hydrolyzed) was determined with a new method in which the steady-state rates of both H(+) ejection and ATP hydrolysis were measured in the presence of K(+) + valinomycin. The H(+)/ATP ratio was found to approach 3.0. A proton cycle for oxidative phosphorylation is proposed, in which four electrochemical H(+) equivalents are ejected per pair of electrons passing each energy-conserving site; three of the H(+) equivalents pass inward to derive ATP synthesis from ADP and phosphate and the fourth H(+) is used to bring about the energy-requiring electrogenic expulsion of ATP(4-) in exchange for extramitochondrial ADP(3-), via the H(+)/H(2)PO(4) (-) symporter. PMID:31621

  9. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    Science.gov (United States)

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

    2003-06-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P proteins (P muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

  10. Synthesis and evaluation of [11C]PyrATP-1, a novel radiotracer for PET imaging of glycogen synthase kinase-3β (GSK-3β)

    International Nuclear Information System (INIS)

    Introduction: The dysfunction of glycogen synthase kinase-3β (GSK-3β) has been implicated in a number of diseases, including Alzheimer’s disease. The ability to non-invasively quantify GSK-3β activity in vivo is therefore of critical importance, and this work is focused upon development of inhibitors of GSK-3β radiolabeled with carbon-11 to examine quantification of the enzyme using positron emission tomography (PET) imaging. Methods: 11C PyrATP-1 was prepared from the corresponding desmethyl-piperazine precursor in an automated synthesis module. In vivo rodent and primate imaging studies were conducted on a Concorde MicroPET P4 scanner to evaluate imaging properties and in vitro autoradiography studies with rat brain samples were carried out to examine specific binding. Results: 2035 ± 518 MBq (55 ± 14 mCi) of [11C]PyrATP-1 was obtained (1%–2% non-corrected radiochemical yield at end-of-synthesis based upon [11C]CO2) with high chemical (> 95%) and radiochemical (> 99%) purities, and good specific activities (143 ± 52 GBq/μmol (3874 ± 1424 Ci/mmol)), n = 5. In vivo microPET imaging studies revealed poor brain uptake in rodents and non-human primates. Pretreatment of rodents with cyclosporin A resulted in moderately increased brain uptake suggesting Pgp transporter involvement. Autoradiography demonstrated high levels of specific binding in areas of the rodent brain known to be rich in GSK-3β. Conclusion: 11C PyrATP-1 is readily synthesized using standard carbon-11 radiochemistry. However the poor brain uptake in rodents and non-human primates indicates that the radiotracer is not suitable for the purposes of quantifying GSK-3β in neurological and psychiatric disorders

  11. Local ATP generation by brain-type creatine kinase (CK-B facilitates cell motility.

    Directory of Open Access Journals (Sweden)

    Jan W P Kuiper

    Full Text Available BACKGROUND: Creatine Kinases (CK catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics. CONCLUSION/SIGNIFICANCE: Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.

  12. Axin is expressed in mitochondria and suppresses mitochondrial ATP synthesis in HeLa cells.

    Science.gov (United States)

    Shin, Jee-Hye; Kim, Hyun-Wook; Rhyu, Im Joo; Kee, Sun-Ho

    2016-01-01

    Many recent studies have revealed that axin is involved in numerous cellular functions beyond the negative regulation of β-catenin-dependent Wnt signaling. Previously, an association of ectopic axin with mitochondria was observed. In an effort to investigate the relationship between axin and mitochondria, we found that axin expression suppressed cellular ATP production, which was more apparent as axin expression levels increased. Also, mitochondrial expression of axin was observed using two axin-expressing HeLa cell models: doxycycline-inducible ectopic axin expression (HeLa-axin) and axin expression enhanced by long-term treatment with XAV939 (HeLa-XAV). In biochemical analysis, axin is associated with oxidative phosphorylation (OXPHOS) complex IV and is involved in defects in the assembly of complex IV-containing supercomplexes. Functionally, axin expression reduced the activity of OXPHOS complex IV and the oxygen consumption rate (OCR), suggesting axin-mediated mitochondrial dysfunction. Subsequent studies using various inhibitors of Wnt signaling showed that the reduction in cellular ATP levels was weaker in cases of ICAT protein expression and treatment with iCRT3 or NSC668036 compared with XAV939 treatment, suggesting that XAV939 treatment affects ATP synthesis in addition to suppressing Wnt signaling activity. Axin-mediated regulation of mitochondrial function may be an additional mechanism to Wnt signaling for regulation of cell growth. PMID:26704260

  13. Synthesis of new technetium brain radiotracers

    International Nuclear Information System (INIS)

    The scintigraphic diagnosis is a major mean for detecting neuro degenerative diseases at early stage; this requires specific radiotracers to a particular class of brain receptors. Our goal was the synthesis of radiotracers, cytectrenes derivatives, which are specific to the 5-HT1A receptor, whose dysfunction is an indicator of neuro degeneration. The study of their biodistribution revealed for only one of them, a good brain retention and a retrieval adequate for diagnosis.

  14. Quantifying ATP turnover in anoxic coleoptiles of rice (Oryza sativa) demonstrates preferential allocation of energy to protein synthesis

    OpenAIRE

    Edwards, Joshua M.; Roberts, Thomas H.; Atwell, Brian J.

    2012-01-01

    Oxygen deprivation limits the energy available for cellular processes and yet no comprehensive ATP budget has been reported for any plant species under O2 deprivation, including Oryza sativa. Using 3-d-old coleoptiles of a cultivar of O. sativa tolerant to flooding at germination, (i) rates of ATP regeneration in coleoptiles grown under normoxia (aerated solution), hypoxia (3% O2), and anoxia (N2) and (ii) rates of synthesis of proteins, lipids, nucleic acids, and cell walls, as well as K+ tr...

  15. Impairment of brain mitochondrial charybdotoxin- and ATP-insensitive BK channel activities in diabetes.

    Science.gov (United States)

    Noursadeghi, E; Jafari, A; Saghiri, R; Sauve, R; Eliassi, A

    2014-12-01

    Existing evidence indicates an impairment of mitochondrial functions and alterations in potassium channel activities in diabetes. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a streptozotocin (STZ) model of diabetes. Using ion channel incorporation of brain mitochondrial inner membrane into the bilayer lipid membrane, we provide in this work evidence for modifications of the mitoBKCa ion permeation properties with channels from vesicles preparations coming from diabetic rats characterized by a significant decrease in conductance. More importantly, the open probability of channels from diabetic rats was reduced 1.5-2.5 fold compared to control, the most significant decrease being observed at depolarizing potentials. Because BKCa β4 subunit has been documented to left shift the BKCa channel voltage dependence curve in high Ca(2+) conditions, a Western blot analysis was undertaken where the expression of mitoBKCa α and β4 subunits was estimated using of anti-α and β4 subunit antibodies. Our results indicated a significant decrease in mitoBKCa β4 subunit expression coupled to a decrease in the expression of α subunit, an observation compatible with the observed decrease in Ca(2+) sensitivity. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from STZ model of diabetic rats, an effect potentially linked to a change in mitoBKCa β4 and α subunits expression and/or to an increase in reactive oxygen species production in high glucose conditions. PMID:25344764

  16. [The effect of magnesium pool isotopy on reactivation of mitochondrial ATP synthesis suppressed by 1-methyl-nicotine amide].

    Science.gov (United States)

    Kuznetsov, D A; Aliautdin, R N; Markarian, A A; Berdieva, A G; Khasigov, P Z; Gatagonova, T M; Ktsoeva, S A; Orlova, M A

    2006-01-01

    The ATP-generating activity of both rat myocardial mitochondria and intramitochondrial creatine phosphokinase (CPK) was examined as a function of the incubation medium magnesium pool isotopy. The in vitro systems tested were prepared from the hearts of animals treated with single injection of 1-methyl-nicotine amide (MNA) suppressing the NAD(P)-dependent reactions in vivo. The presense of the 25Mg paramagnetic cations leads to essential compensation of intramitochondrial ATP deficiency caused by the MNA induced blockade of oxidative phosphorylation. This effect is merely unreachable in those systems where the magnesium pool consists of isotopes with a zero nuclear spin (24Mg, 26Mg). The reactivation of mitochondrial ATP synthesis described here involves CPK activity which does not depends on MNA. In this case, a high efficiency of this reactivation seems to be a spin selective phenomenon which requires, predominantly, 25Mg2+ cations. PMID:16805385

  17. An Ancient Pathway Combining Carbon Dioxide Fixation with the Generation and Utilization of a Sodium Ion Gradient for ATP Synthesis

    Science.gov (United States)

    Poehlein, Anja; Schmidt, Silke; Kaster, Anne-Kristin; Goenrich, Meike; Vollmers, John; Thürmer, Andrea; Bertsch, Johannes; Schuchmann, Kai; Voigt, Birgit; Hecker, Michael; Daniel, Rolf; Thauer, Rudolf K.; Gottschalk, Gerhard; Müller, Volker

    2012-01-01

    Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes. PMID:22479398

  18. Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury.

    Directory of Open Access Journals (Sweden)

    Hey-Kyeong Jeong

    Full Text Available BACKGROUND: Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. METHODS AND FINDINGS: Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH(+ and Iba-1(+ cells acutely died, and as the penumbra the area surrounding the core where Iba-1(+ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. CONCLUSIONS: Different cellular

  19. Growth enhancement effect of BzATP on primary cultured astrocytes from rat brain

    Institute of Scientific and Technical Information of China (English)

    Hua-Zheng LIANG; Ying LIU; Zhu-Rong YE

    2006-01-01

    Objective To explore whether BzATP could promote the growth of primary cultured astrocytes (AS) of rat and its possible mechanism, and whether TGF-β1 was involved in the event. Methods The primary cultured AS were derived from new born Sprague-Dawley rats.Glial fibrillary acidic protein (GFAP) immunofluorescent stain was used to check the purity of cultured AS. Morphometry was used to detect the changes of AS. The proliferation index of AS was detected by BrdU incorporation assay. Western blot was used to detect the changes of GFAP under different conditions. Changes of TGF-β1 gene transcription were detected by RT-PCR. ELISA was utilized to detect the variation of TGF-β1 protein in the supernate. Results The purity of primary cultured AS reached to 99%. BzATP promoted the hypertrophy of AS including the elongation of AS processes and the enlargement of cell bodies, BzATP also promoted the expression of GFAP in existence of Ca2+, but had no effect on cell proliferation. BzATP increased the transcription of TGF-β1 mRNA and the release of TGF-β1 protein in existence of Ca2+. TGF-β1 neutralizing antibody partially inhibited the expression of GFAP induced by BzATP, but had no effect on AS proliferation and cell morphology. Conclusion BzATP enhanced the hypertrophy of primary cultured AS, increased the expression of GFAP partially through TGF-β1. Mechanisms of the enhancement of AS growth induced by BzATP other than TGF-51 pathway remains to be elucidated.

  20. Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function.

    Science.gov (United States)

    van den Broek, N M A; Ciapaite, J; Nicolay, K; Prompers, J J

    2010-11-01

    (31)P magnetic resonance spectroscopy (MRS) has been used to assess skeletal muscle mitochondrial function in vivo by measuring 1) phosphocreatine (PCr) recovery after exercise or 2) resting ATP synthesis flux with saturation transfer (ST). In this study, we compared both parameters in a rat model of mitochondrial dysfunction with the aim of establishing the most appropriate method for the assessment of in vivo muscle mitochondrial function. Mitochondrial dysfunction was induced in adult Wistar rats by daily subcutaneous injections with the complex I inhibitor diphenyleneiodonium (DPI) for 2 wk. In vivo (31)P MRS measurements were supplemented by in vitro measurements of oxygen consumption in isolated mitochondria. Two weeks of DPI treatment induced mitochondrial dysfunction, as evidenced by a 20% lower maximal ADP-stimulated oxygen consumption rate in isolated mitochondria from DPI-treated rats oxidizing pyruvate plus malate. This was paralleled by a 46% decrease in in vivo oxidative capacity, determined from postexercise PCr recovery. Interestingly, no significant difference in resting, ST-based ATP synthesis flux was observed between DPI-treated rats and controls. These results show that PCr recovery after exercise has a more direct relationship with skeletal muscle mitochondrial function than the ATP synthesis flux measured with (31)P ST MRS in the resting state. PMID:20668212

  1. A mechano-chemiosmotic model for the coupling of electron and proton transfer to ATP synthesis in energy-transforming membranes: a personal perspective

    OpenAIRE

    Kasumov, Eldar A.; Kasumov, Ruslan E.; Kasumova, Irina V.

    2014-01-01

    ATP is synthesized using ATP synthase by utilizing energy either from the oxidation of organic compounds, or from light, via redox reactions (oxidative- or photo phosphorylation), in energy-transforming membranes of mitochondria, chloroplasts, and bacteria. ATP synthase undergoes several changes during its functioning. The generally accepted model for ATP synthesis is the well-known rotatory model (see e.g., Junge et al., Nature 459:364–370, 2009; Junge and Müller, Science 333:704–705, 2011)....

  2. Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity

    Indian Academy of Sciences (India)

    Haseeb Ahmad Khan

    2003-06-01

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the analysis of ATP in small tissue samples. The results showed that the sensitivity of the FLB test can be enhanced several fold by using ultraturax homogenizer, perchloric acid extraction, neutralization of acid extract and its optimal dilution, before performing the assay reaction.

  3. Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells.

    Science.gov (United States)

    Peyta, Laure; Jarnouen, Kathleen; Pinault, Michelle; Guimaraes, Cyrille; Pais de Barros, Jean-Paul; Chevalier, Stephan; Dumas, Jean-François; Maillot, François; Hatch, Grant M; Loyer, Pascal; Servais, Stephane

    2016-04-01

    Cardiolipin (CL) is a unique mitochondrial phospholipid potentially affecting many aspects of mitochondrial function/processes, i.e. energy production through oxidative phosphorylation. Most data focusing on implication of CL content and mitochondrial bioenergetics were performed in yeast or in cellular models of Barth syndrome. Previous work reported that increase in CL content leads to decrease in liver mitochondrial ATP synthesis yield. Therefore the aim of this study was to determine the effects of moderate decrease in CL content on mitochondrial bioenergetics in human hepatocytes. For this purpose, we generated a cardiolipin synthase knockdown (shCLS) in HepaRG hepatoma cells showing bioenergetics features similar to primary human hepatocytes. shCLS cells exhibited a 55% reduction in CLS gene and a 40% decrease in protein expression resulting in a 45% lower content in CL compared to control (shCTL) cells. Oxygen consumption was significantly reduced in shCLS cells compared to shCTL regardless of substrate used and energy state analyzed. Mitochondrial low molecular weight supercomplex content was higher in shCLS cells (+60%) compared to shCTL. Significant fragmentation of the mitochondrial network was observed in shCLS cells compared to shCTL cells. Surprisingly, mitochondrial ATP synthesis was unchanged in shCLS compared to shCTL cells but exhibited a higher ATP:O ratio (+46%) in shCLS cells. Our results suggest that lowered respiratory chain activity induced by moderate reduction in CL content may be due to both destabilization of supercomplexes and mitochondrial network fragmentation. In addition, CL content may regulate mitochondrial ATP synthesis yield. PMID:26768115

  4. In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier.

    Science.gov (United States)

    Bakhsheshian, Joshua; Wei, Bih-Rong; Hall, Matthew D; Simpson, R Mark; Gottesman, Michael M

    2016-01-01

    We provide a detailed protocol for imaging ATP-binding cassette subfamily G member 2 (ABCG2) function at the blood-brain barrier (BBB) of transgenic mice. D-Luciferin is specifically transported by ABCG2 found on the apical side of endothelial cells at the BBB. The luciferase-luciferin enzymatic reaction produces bioluminescence, which allows a direct measurement of ABCG2 function at the BBB. Therefore bioluminescence imaging (BLI) correlates with ABCG2 function at the BBB and this can be measured by administering luciferin in a mouse model that expresses luciferase in the brain parenchyma. BLI allows for a relatively low-cost alternative for studying transporter function in vivo compared to other strategies such as positron emission tomography. This method for imaging ABCG2 function at the BBB can be used to investigate pharmacokinetic inhibition of the transporter. PMID:27424909

  5. Prenatal Ethanol Exposure Up-Regulates the Cholesterol Transporters ATP-Binding Cassette A1 and G1 and Reduces Cholesterol Levels in the Developing Rat Brain

    OpenAIRE

    Zhou, Chunyan; Chen, Jing; Zhang, Xiaolu; Costa, Lucio G.; Guizzetti, Marina

    2014-01-01

    Aims: Cholesterol plays a pivotal role in many aspects of brain development; reduced cholesterol levels during brain development, as a consequence of genetic defects in cholesterol biosynthesis, leads to severe brain damage, including microcephaly and mental retardation, both of which are also hallmarks of the fetal alcohol syndrome. We had previously shown that ethanol up-regulates the levels of two cholesterol transporters, ABCA1 (ATP binding cassette-A1) and ABCG1, leading to increased cho...

  6. Two strategies for the synthesis of the biologically important ATP analogue ApppI, at a multi-milligram scale

    Directory of Open Access Journals (Sweden)

    Janne Weisell

    2015-11-01

    Full Text Available Two strategies for the synthesis of the ATP (adenosine triphosphate analogue ApppI [1-adenosin-5’-yl 3-(3-methylbut-3-enyltriphosphoric acid diester] (1 are described. ApppI is an active metabolite of the mevalonate pathway and thus is of major biological significance. Chemically synthezised ApppI was purified by using triethylammonium bicarbonate as the counter ion in ion-pair chromatography and characterized by 1H, 13C, 31P NMR and MS spectroscopical methods.

  7. Two strategies for the synthesis of the biologically important ATP analogue ApppI, at a multi-milligram scale

    Science.gov (United States)

    Weisell, Janne; Vepsäläinen, Jouko

    2015-01-01

    Summary Two strategies for the synthesis of the ATP (adenosine triphosphate) analogue ApppI [1-adenosin-5’-yl 3-(3-methylbut-3-enyl)triphosphoric acid diester] (1) are described. ApppI is an active metabolite of the mevalonate pathway and thus is of major biological significance. Chemically synthezised ApppI was purified by using triethylammonium bicarbonate as the counter ion in ion-pair chromatography and characterized by 1H, 13C, 31P NMR and MS spectroscopical methods. PMID:26664641

  8. ATP synthesis at 100 degrees C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi.

    Science.gov (United States)

    Dirmeier, R; Hauska, G; Stetter, K O

    2000-02-01

    The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100 degrees C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF(1) headpiece, and the 7 kDa proteolipid of the AF(0) component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 micromol per min and mg protein) as well as ATP formation (up to 0.4 micromol per min and mg membrane protein) were highest at 100 degrees C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of (33)P(i)-labeled ADP and of another compound not identified so far. PMID:10664465

  9. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania.

    Science.gov (United States)

    Luque-Ortega, Juan Román; van't Hof, Wim; Veerman, Enno C I; Saugar, José M; Rivas, Luis

    2008-06-01

    Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed. PMID:18230684

  10. Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis, and ATP production in human skeletal muscle without amino acid replacement

    OpenAIRE

    Barazzoni, Rocco; Short, Kevin R.; Asmann, Yan; Coenen-Schimke, Jill M.; Robinson, Matthew M.; Sreekumaran Nair, K.

    2012-01-01

    Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-13C]leucine as a tracer, we measured the fractional synthetic rate of mitochondri...

  11. A mechano-chemiosmotic model for the coupling of electron and proton transfer to ATP synthesis in energy-transforming membranes: a personal perspective.

    Science.gov (United States)

    Kasumov, Eldar A; Kasumov, Ruslan E; Kasumova, Irina V

    2015-01-01

    ATP is synthesized using ATP synthase by utilizing energy either from the oxidation of organic compounds, or from light, via redox reactions (oxidative- or photo phosphorylation), in energy-transforming membranes of mitochondria, chloroplasts, and bacteria. ATP synthase undergoes several changes during its functioning. The generally accepted model for ATP synthesis is the well-known rotatory model (see e.g., Junge et al., Nature 459:364-370, 2009; Junge and Müller, Science 333:704-705, 2011). Here, we present an alternative modified model for the coupling of electron and proton transfer to ATP synthesis, which was initially developed by Albert Lester Lehninger (1917-1986). Details of the molecular mechanism of ATP synthesis are described here that involves cyclic low-amplitude shrinkage and swelling of mitochondria. A comparison of the well-known current model and the mechano-chemiosmotic model is also presented. Based on structural, and other data, we suggest that ATP synthase is a Ca(2+)/H(+)-K(+) Cl(-)-pump-pore-enzyme complex, in which γ-subunit rotates 360° in steps of 30°, and 90° due to the binding of phosphate ions to positively charged amino acid residues in the N-terminal γ-subunit, while in the electric field. The coiled coil b 2-subunits are suggested to act as ropes that are shortened by binding of phosphate ions to positively charged lysines or arginines; this process is suggested to pull the α 3 β 3-hexamer to the membrane during the energization process. ATP is then synthesized during the reverse rotation of the γ-subunit by destabilizing the phosphated N-terminal γ-subunit and b 2-subunits under the influence of Ca(2+) ions, which are pumped over from storage-intermembrane space into the matrix, during swelling of intermembrane space. In the process of ATP synthesis, energy is first, predominantly, used in the delivery of phosphate ions and protons to the α 3 β 3-hexamer against the energy barrier with the help of C-terminal alpha

  12. Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis.

    OpenAIRE

    Kooistra, J; Venema, G

    1991-01-01

    The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA pro...

  13. Alkaliphilic bacteria with impact on industrial applications, concepts of early life forms and bioenergetics of ATP synthesis

    Directory of Open Access Journals (Sweden)

    Laura ePreiss

    2015-06-01

    Full Text Available Alkaliphilic bacteria typically grow well at pH 9, with the most extremophilic strains growing up to pH values as high as pH 12-13. Interest in extreme alkaliphiles arises because they are sources of useful, stable enzymes, and the cells themselves can be used for biotechnological and other applications at high pH. In addition, alkaline hydrothermal vents represent an early evolutionary niche for alkaliphiles and novel extreme alkaliphiles have also recently been found in alkaline serpentinizing sites. A third focus of interest in alkaliphiles is the challenge raised by the use of proton-coupled ATP synthases for oxidative phosphorylation by non-fermentative alkaliphiles. This creates a problem with respect to tenets of the chemiosmotic model that remains the core model for the bioenergetics of oxidative phosphorylation. Each of these facets of alkaliphilic bacteria will be discussed with a focus on extremely alkaliphilic Bacillus strains. These alkaliphilic bacteria have provided a cogent experimental system to probe adaptations that enable their growth and oxidative phosphorylation at high pH. Adaptations are clearly needed to enable secreted or partially exposed enzymes or protein complexes to function at the high external pH. Also, alkaliphiles must maintain a cytoplasmic pH that is significantly lower than the pH of the outside medium. This protects cytoplasmic components from an external pH that is alkaline enough to impair their stability or function. However, the pH gradient across the cytoplasmic membrane, with its orientation of more acidic inside than outside, is in the reverse of the productive orientation for bioenergetic work. The reversed gradient reduces the trans-membrane proton motive force available to energize ATP synthesis. Multiple strategies are hypothesized to be involved in enabling alkaliphiles to circumvent the challenge of a low bulk proton-motive force energizing proton-coupled ATP synthesis at high pH.

  14. Alkaliphilic Bacteria with Impact on Industrial Applications, Concepts of Early Life Forms, and Bioenergetics of ATP Synthesis.

    Science.gov (United States)

    Preiss, Laura; Hicks, David B; Suzuki, Shino; Meier, Thomas; Krulwich, Terry Ann

    2015-01-01

    Alkaliphilic bacteria typically grow well at pH 9, with the most extremophilic strains growing up to pH values as high as pH 12-13. Interest in extreme alkaliphiles arises because they are sources of useful, stable enzymes, and the cells themselves can be used for biotechnological and other applications at high pH. In addition, alkaline hydrothermal vents represent an early evolutionary niche for alkaliphiles and novel extreme alkaliphiles have also recently been found in alkaline serpentinizing sites. A third focus of interest in alkaliphiles is the challenge raised by the use of proton-coupled ATP synthases for oxidative phosphorylation by non-fermentative alkaliphiles. This creates a problem with respect to tenets of the chemiosmotic model that remains the core model for the bioenergetics of oxidative phosphorylation. Each of these facets of alkaliphilic bacteria will be discussed with a focus on extremely alkaliphilic Bacillus strains. These alkaliphilic bacteria have provided a cogent experimental system to probe adaptations that enable their growth and oxidative phosphorylation at high pH. Adaptations are clearly needed to enable secreted or partially exposed enzymes or protein complexes to function at the high external pH. Also, alkaliphiles must maintain a cytoplasmic pH that is significantly lower than the pH of the outside medium. This protects cytoplasmic components from an external pH that is alkaline enough to impair their stability or function. However, the pH gradient across the cytoplasmic membrane, with its orientation of more acidic inside than outside, is in the reverse of the productive orientation for bioenergetic work. The reversed gradient reduces the trans-membrane proton-motive force available to energize ATP synthesis. Multiple strategies are hypothesized to be involved in enabling alkaliphiles to circumvent the challenge of a low bulk proton-motive force energizing proton-coupled ATP synthesis at high pH. PMID:26090360

  15. Synthesis, characterization, and in vitro cytotoxic effects of K4 (PtCl2ATP)

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, K.K.; Bhattacharyya, R.; Maity, P. (Jadavpur Univ. Calcutta (India))

    1991-03-01

    An antineoplastic agent, cis-K4 (PtCl2ATP) has been synthesized and characterized, using elemental analysis, solution conductance, thermoanalysis, infrared, NMR spectroscopy, and circular dichroism studies. The in vitro cytotoxic effect imparted by this compound on Dalton's Lymphoma cells has been assessed by Trypan blue dye exclusion method and 51Cr release assays.

  16. Synthesis, characterization, and in vitro cytotoxic effects of K4 [PtCl2ATP

    International Nuclear Information System (INIS)

    An antineoplastic agent, cis-K4 [PtCl2ATP] has been synthesized and characterized, using elemental analysis, solution conductance, thermoanalysis, infrared, NMR spectroscopy, and circular dichroism studies. The in vitro cytotoxic effect imparted by this compound on Dalton's Lymphoma cells has been assessed by Trypan blue dye exclusion method and 51Cr release assays

  17. A metabolic link between mitochondrial ATP synthesis and liver glycogen metabolism: NMR study in rats re-fed with butyrate and/or glucose

    Directory of Open Access Journals (Sweden)

    Beauvieux Marie-Christine

    2011-06-01

    Full Text Available Abstract Background Butyrate, end-product of intestinal fermentation, is known to impair oxidative phosphorylation in rat liver and could disturb glycogen synthesis depending on the ATP supplied by mitochondrial oxidative phosphorylation and cytosolic glycolysis. Methods In 48 hr-fasting rats, hepatic changes of glycogen and total ATP contents and unidirectional flux of mitochondrial ATP synthesis were evaluated by ex vivo 31P NMR immediately after perfusion and isolation of liver, from 0 to 10 hours after force-feeding with (butyrate 1.90 mg + glucose 14.0 mg.g-1 body weight or isocaloric glucose (18.2 mg.g-1 bw; measurements reflected in vivo situation at each time of liver excision. The contribution of energetic metabolism to glycogen metabolism was estimated. Results A net linear flux of glycogen synthesis (~11.10 ± 0.60 μmol glucosyl units.h-1.g-1 liver wet weight occurred until the 6th hr post-feeding in both groups, whereas butyrate delayed it until the 8th hr. A linear correlation between total ATP and glycogen contents was obtained (r2 = 0.99 only during net glycogen synthesis. Mitochondrial ATP turnover, calculated after specific inhibition of glycolysis, was stable (~0.70 ± 0.25 μmol.min-1.g-1 liver ww during the first two hr whatever the force-feeding, and increased transiently about two-fold at the 3rd hr in glucose. Butyrate delayed the transient increase (1.80 ± 0.33 μmol.min-1.g-1 liver ww to the 6th hr post-feeding. Net glycogenolysis always appeared after the 8th hr, whereas flux of mitochondrial ATP synthesis returned to near basal level (0.91 ± 0.19 μmol.min-1.g-1 liver ww. Conclusion In liver from 48 hr-starved rats, the energy need for net glycogen synthesis from exogenous glucose corresponds to ~50% of basal mitochondrial ATP turnover. The evidence of a late and transient increase in mitochondrial ATP turnover reflects an energetic need, probably linked to a glycogen cycling. Butyrate, known to reduce oxidative

  18. Multiple sites of vasopressin synthesis in the injured brain

    OpenAIRE

    Szmydynger-Chodobska, Joanna; Zink, Brian J.; Chodobski, Adam

    2010-01-01

    Previous studies have indicated that the primary targets for vasopressin actions on the injured brain are the cerebrovascular endothelium and astrocytes, and that vasopressin amplifies the posttraumatic production of proinflammatory mediators. Here, the controlled cortical impact model of traumatic brain injury in rats was used to identify the sources of vasopressin in the injured brain. Injury increased vasopressin synthesis in the hypothalamus and cerebral cortex adjacent to the posttraumat...

  19. Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.; Robertson, D.W. (Eli Lilly and Co., Indianapolis, IN (USA))

    1991-05-01

    We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.

  20. Quantitative autoradiography of the binding sites for [125I] iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

    International Nuclear Information System (INIS)

    We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with [125I]iodoglyburide (N-[2-[[[(cyclohexylamino)carbonyl]amino]sulfonyl]ethyl]-5-125I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific [125I]iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of [125I]iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels

  1. The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

    Directory of Open Access Journals (Sweden)

    Sunil Nath

    2008-09-01

    Full Text Available Complete details of the thermodynamics and molecular mechanisms of ATP synthesis/hydrolysis and muscle contraction are offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT energy storage mechanism of muscle contraction. The manifold fundamental consequences and mechanistic implications of the unified theory for oxidative phosphorylation and muscle contraction are explained. The consistency of current mechanisms of ATP synthesis and muscle contraction with experiment is assessed, and the novel insights of the unified theory are shown to take us beyond the binding change mechanism, the chemiosmotic theory and the lever arm model. It is shown from first principles how previous theories of ATP synthesis and muscle contraction violate both the first and second laws of thermodynamics, necessitating their revision. It is concluded that the new paradigm, ten years after making its first appearance, is now perfectly poised to replace the older theories. Finally, applications of the unified theory in cell life and cell death are outlined and prospects for future research are explored. While it is impossible to cover each and every specific aspect of the above, an attempt has been made here to address all the pertinent details and what is presented should be sufficient to convince the reader of the novelty, originality, breakthrough nature and power of the unified theory, its manifold fundamental consequences and mechanistic implications, and its applications in health and disease.

  2. Translational coupling of the maize chloroplast atpB and atpE genes

    OpenAIRE

    Gatenby, Anthony A.; Rothstein, Steven. J.; Nomura, Masayasu

    1989-01-01

    The genes for the β and ε subunits of maize chloroplast ATP synthase are encoded by the organelle genome, are cotranscribed, and have overlapping translation initiation and termination codons. To determine whether the atpB and atpE genes are translationally coupled, they were transformed into Escherichia coli on a multicopy plasmid. Synthesis of full-length β and ε polypeptides demonstrated correct initiation of translation by the bacterial ribosomes. To assay for translational coupling, the ...

  3. Direct and simultaneous measurements of light-driven pH gradient and ATP synthesis by 31P-NMR for the chromatophores of Rhodopseudomonas spheroides G1C

    International Nuclear Information System (INIS)

    The correlation between ATP synthesis and light-driven pH gradient across the chromatophore membranes of Rhodopseudomonas spheroides G1C was investigated employing 31P-NMR. The internal and external pH were determined by measuring the chemical shift of inorganic phosphate inside and outside the chromatophores, respectively. When the chromatophore suspension was illuminated in the presence of ADP outside the chromatophores, generation of the pH gradient and ATP synthesis could be detected simultaneously. The authors recorded the NMR spectra changing with illumination time, initial external pH and light intensity. The apparent rate of ATP synthesis was determined from the intensities of the signals ascribed to ADP and ATP. The rate of ATP synthesis during the first 30 min illumination was compared with ΔpH generated. The ATP synthesis started at ΔpH ∼ 0.5 and increased its rate with the increase of ΔpH. The rate was almost saturated at above ΔpH ∼ 1.5 or external pH ∼ 7.2. Therefore it can be said that under continuous illumination, the photophosphorylation system of this chromatophore works most efficiently in the physiological pH range. The apparent rate of ATP synthesis was found to decrease at higher external pH and for the illuminations longer than 30 min. This was attributed to ATP hydrolysis which was more efficient at a higher pH. (Auth.)

  4. TSH increases synthesis of hepatic ATP-binding cassette subfamily A member 1 in hypercholesterolemia.

    Science.gov (United States)

    Zhang, Tiantian; Zhou, Lingyan; Li, Cong Cong; Shi, Hong; Zhou, Xinli

    2016-07-22

    Epidemiological evidence suggests that thyrotropin (TSH) levels are closely correlated with the severity of hypercholesterolemia. Reverse cholesterol transfer (RCT) plays an important role in regulating bloodcholesterol. However, the molecular mechanism of hypercholesterolemia in subclinical hypothyroidism (SCH) has not been fully clarified. The SCH mouse model, which is characterized by elevated serum TSH but not thyroid hormone levels, demonstrated a significant increase in plasma cholesterol compared with controls. Interestingly, Tshr KO mice, with normal thyroid hormone levels after thyroid hormone supplementation, showed lower plasma cholesterol levels compared with their wild-type littermates. ATP binding cassette subfamily A member 1(ABCA1) is a member of the ABC superfamily, which induces transfer of intracellular cholesterol to extracellular apolipoprotein. TSH upregulated hepatic ABCA1 to promote the efflux of intercellular cumulative cholesterol, resulting in increased plasma cholesterol. These data might partially explain the pathogenesis of hypercholesterolemia in SCH. PMID:27179782

  5. The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth.

    Science.gov (United States)

    Karlsson, Patrik M; Herdean, Andrei; Adolfsson, Lisa; Beebo, Azeez; Nziengui, Hugues; Irigoyen, Sonia; Ünnep, Renáta; Zsiros, Ottó; Nagy, Gergely; Garab, Győző; Aronsson, Henrik; Versaw, Wayne K; Spetea, Cornelia

    2015-10-01

    The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high-phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO2 fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non-photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton-motive force across thylakoids. Small-angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long-range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild-type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro-organization of complexes and induction of photoprotective mechanisms. PMID:26255788

  6. SHORT-TERM MEMORY IS INDEPENDENT OF BRAIN PROTEIN SYNTHESIS

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Hasker P.; Rosenzweig, Mark R.; Jones, Oliver W.

    1980-09-01

    Male Swiss albino CD-1 mice given a single injection of a cerebral protein synthesis inhibitor, anisomycin (ANI) (1 mg/animal), 20 min prior to single trial passive avoidance training demonstrated impaired retention at tests given 3 hr, 6 hr, 1 day, and 7 days after training. Retention was not significantly different from saline controls when tests were given 0.5 or 1.5 hr after training. Prolonging inhibition of brain protein synthesis by giving either 1 or 2 additional injections of ANI 2 or 2 and 4 hr after training did not prolong short-term retention performance. The temporal development of impaired retention in ANI treated mice could not be accounted for by drug dosage, duration of protein synthesis inhibition, or nonspecific sickness at test. In contrast to the suggestion that protein synthesis inhibition prolongs short-term memory (Quinton, 1978), the results of this experiment indicate that short-term memory is not prolonged by antibiotic drugs that inhibit cerebral protein synthesis. All evidence seems consistent with the hypothesis that short-term memory is protein synthesis independent and that the establishment of long-term memory depends upon protein synthesis during or shortly after training. Evidence for a role of protein synthesis in memory maintenance is discussed.

  7. Synthesis of a new technetium brain radiotracer

    International Nuclear Information System (INIS)

    The radiological diagnosis of brain diseases, including neurodegenerative ones, is still difficult because of the absence of specific biological markers. These diseases are then increasingly the subject of researches and new experiments. The biodistribution of a new cytectrenes-aniline derivative, having a lipophilic character and low molecular weight, showed a prolonged brain retention and a specific tissue distribution of the hippocampus. This radiotracer could contribute to establish early diagnosis of neurodegenerative diseases thanks to its affinity with a serotonin receptors-rich region. (Author)

  8. Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

    Science.gov (United States)

    Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

    2015-09-01

    The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica. PMID:25959598

  9. Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

    OpenAIRE

    Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Nair, K. Sreekumaran

    2003-01-01

    Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32–42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP product...

  10. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb’s cycle flux independent of ATP synthesis

    International Nuclear Information System (INIS)

    Highlights: ► We studied media effects on mechanisms of insulin secretion of INS-1 cells. ► Insulin secretion was higher in DMEM than KRB despite identical ATP synthesis rates. ► Insulin secretion rates correlated with rates of anaplerosis and TCA cycle. ► Mitochondria metabolism and substrate cycles augment secretion signal of ATP. -- Abstract: Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with 31P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by 13C NMR isotopomer analysis of the fate of [U-13C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was

  11. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb's cycle flux independent of ATP synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Cline, Gary W., E-mail: gary.cline@yale.edu [The Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520 (United States); Department of Surgery, University of Minnesota-Twin Cities, Minneapolis, MN 55455 (United States); Pongratz, Rebecca L.; Zhao, Xiaojian [The Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520 (United States); Papas, Klearchos K. [Department of Surgery, University of Minnesota-Twin Cities, Minneapolis, MN 55455 (United States)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer We studied media effects on mechanisms of insulin secretion of INS-1 cells. Black-Right-Pointing-Pointer Insulin secretion was higher in DMEM than KRB despite identical ATP synthesis rates. Black-Right-Pointing-Pointer Insulin secretion rates correlated with rates of anaplerosis and TCA cycle. Black-Right-Pointing-Pointer Mitochondria metabolism and substrate cycles augment secretion signal of ATP. -- Abstract: Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with {sup 31}P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by {sup 13}C NMR isotopomer analysis of the fate of [U-{sup 13}C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found

  12. A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation

    Directory of Open Access Journals (Sweden)

    Sikorska Marianna

    2008-02-01

    Full Text Available Abstract Background Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD. These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells. Results RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis. Conclusion Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

  13. The impact of mitochondrial tRNA mutations on the amount of ATP synthase differs in the brain compared to other tissues.

    Science.gov (United States)

    Fornuskova, Daniela; Brantova, Olga; Tesarova, Marketa; Stiburek, Lukas; Honzik, Tomas; Wenchich, Laszlo; Tietzeova, Evzenie; Hansikova, Hana; Zeman, Jiri

    2008-05-01

    The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNA(Lys) (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNA(LeuUUR). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits. PMID:18319067

  14. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb's cycle flux independent of ATP synthesis.

    Science.gov (United States)

    Cline, Gary W; Pongratz, Rebecca L; Zhao, Xiaojian; Papas, Klearchos K

    2011-11-11

    Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with (31)P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by (13)C NMR isotopomer analysis of the fate of [U-(13)C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media. PMID:22008547

  15. Defects in mitochondrial ATP synthesis in dystrophin-deficient mdx skeletal muscles may be caused by complex I insufficiency.

    Directory of Open Access Journals (Sweden)

    Emma Rybalka

    Full Text Available Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca2+ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca2+ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca2+ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb's cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca2+ load in comparison to controls, and 400 nM extramitochondrial Ca2+ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.

  16. [31P-NMR analysis of high energy phosphorous compounds (ATP and phosphocreatine) in the living rat brain--effects of halothane anesthesia and a hypoxic condition].

    Science.gov (United States)

    Yuasa, T; Miyatake, T; Kuwabara, T; Umeda, M; Eguchi, K

    1983-11-01

    31phosphorus nuclear magnetic resonance (31P-NMR) measurements have provided new and valuable insights for studying the metabolism of living systems. The aim of this paper is to introduce a technique of application of 31P-NMR measurements using a surface coil method, and to discuss the effects of halothane anesthesia and hypoxic hypoxia on the energetic metabolism of intact rat brains. All measurements were made using a JEOL FX 270 spectrometer with a super conducting magnet of 54-mm bore diameter. The magnetic field intensity of this machine is 6.3 tesla, and the resonance frequency used for 31P was 109.14 MHz. We remodelled an ordinary probe to take a live rat, and the animals were made to inhale anesthetic halothane or mixture of oxygen and nitrogen at various concentrations controlled by a flow regulator. The best conditions for measurements with our surface coil method were determined in this study as follows: (1) 90 degrees pulse width and selectivity, Fig. 1 shows signal selectivity in depthwise direction changed with 90 degrees pulse width, which was set to 20 microseconds. (2) Sensitivity and resolution; To obtain a spectrum of 31P-NMR from a rat brain 500 accumulations of free induction decays were considered suitable for both time and space resolution. Fig. 2 shows variations of signal intensity with pulse repetition time, which was set to 2 sec. It took about 17 min for averaging to get a spectrogram. (3) Quantitative accuracy and qualification; As shown in Fig. 3, a linear relationship was found between the signal intensity of beta-phosphate of ATP and the concentration of ATP solutions, thus proving the quantitative accuracy of our systems.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6661335

  17. Duchenne muscular dystrophy: normal ATP turnover in cultured cells

    International Nuclear Information System (INIS)

    This paper examines ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients and there was no difference in ATP synthesis or degradation. The ATP synthesis was measured by the incorporation of C 14-U-adenine into aTP and ADP. although there was a significant decrease in radioactively labelled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration of ADP metabolism

  18. FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity.

    Science.gov (United States)

    Gavini, N; Burgess, B K

    1992-10-15

    We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein. PMID:1400428

  19. Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscles May Be Caused by Complex I Insufficiency

    OpenAIRE

    Rybalka, Emma; Cara A. Timpani; Cooke, Matthew B; Williams, Andrew D.; Hayes, Alan

    2014-01-01

    Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca2+ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca2+ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted ...

  20. Diminished synthesis of subunit a (ATP6) and altered function of ATP synthase and cytochrome c oxidase due to the mtDNA 2 bp microdeletion of TA at positions 9205 and 9206

    Czech Academy of Sciences Publication Activity Database

    Ješina, Pavel; Tesařová, M.; Fornůsková, D.; Vojtíšková, Alena; Pecina, Petr; Kaplanová, Vilma; Hansíková, H.; Zeman, J.; Houštěk, Josef

    2004-01-01

    Roč. 383, č. 3 (2004), s. 561-571. ISSN 0264-6021 R&D Projects: GA ČR(CZ) GA303/03/0749; GA MZd(CZ) NR7790; GA MZd(CZ) NR8065 Grant ostatní: GA UK(CZ) 14/2004 Institutional research plan: CEZ:AV0Z5011922 Keywords : ATP6 * ATP synthase * mitochondrial disease Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.278, year: 2004

  1. Design, Synthesis, and Identification of Silicon Incorporated Oxazolidinone Antibiotics with Improved Brain Exposure.

    Science.gov (United States)

    Seetharamsingh, B; Ramesh, Remya; Dange, Santoshkumar S; Khairnar, Pankaj V; Singhal, Smita; Upadhyay, Dilip; Veeraraghavan, Sridhar; Viswanadha, Srikant; Vakkalanka, Swaroop; Reddy, D Srinivasa

    2015-11-12

    Therapeutic options for brain infections caused by pathogens with a reduced sensitivity to drugs are limited. Recent reports on the potential use of linezolid in treating brain infections prompted us to design novel compounds around this scaffold. Herein, we describe the design and synthesis of various oxazolidinone antibiotics with the incorporation of silicon. Our findings in preclinical species suggest that silicon incorporation is highly useful in improving brain exposures. Interestingly, three compounds from this series demonstrated up to a 30-fold higher brain/plasma ratio when compared to linezolid thereby indicating their therapeutic potential in brain associated disorders. PMID:26617962

  2. Astrocytes release ATP through lysosomal exocytosis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Astrocytes, the most abundant type of glial cells in the brain, have been found to release signaling molecules, including adenosine triphosphate(ATP), the most important energy carrier inside the cell as well as a universal extracellular signaling molecule.

  3. Response of rat brain protein synthesis to ethanol and sodium barbital

    International Nuclear Information System (INIS)

    Central nervous system (CNS) depressants such as ethanol and barbiturates under acute or chronic conditions can induce changes in rat brain protein synthesis. While these data demonstrate the individual effects of drugs on protein synthesis, the response of brain protein synthesis to alcohol-drug interactions is not known. The goal of the present study was to determine the individual and combined effects of ethanol and sodium barbital on brain protein synthesis and gain an understanding of the mechanisms by which these alterations in protein synthesis are produced. Specifically, the in vivo and in vitro effects of sodium barbital (one class of barbiturates which is not metabolized by the hepatic tissue) were examined on brain protein synthesis in rats made physically dependent upon ethanol. Using cell free brain polysomal systems isolated from Control, Ethanol and 24 h Ethanol Withdrawn rats, data show that sodium barbital, when intubated intragastrically, inhibited the time dependent incorporation of 14C) leucine into protein by all three groups of ribosomes. Under these conditions, the Ethanol Withdrawn group displayed the largest inhibition of the 14C) leucine incorporation into protein when compared to the Control and Ethanol groups. In addition, sodium barbital when added at various concentrations in vitro to the incubation medium inhibited the incorporation of 14C) leucine into protein by Control and Ethanol polysomes. The inhibitory effects were also obtained following preincubation of ribosomes in the presence of barbital but not cycloheximide. Data suggest that brain protein synthesis, specifically brain polysomes, through interaction with ethanol or barbital are involved in the functional development of tolerance. These interactions may occur through proteins or polypeptide chains or alterations in messenger RNA components associated with the ribosomal units

  4. Optical ATP Biosensor for Extracellular ATP Measurement

    OpenAIRE

    Wang, C; Huang, C.-Y.C.; Lin, W-C

    2013-01-01

    Extracellular Adenosine-5′-triphosphate (ATP) is an important multi-functional molecule which can mediate numerous physiological activities by activating purinergic P2 receptors. The objective of this study was to develop a novel optical ATP sensor for in-situ extracellular ATP measurement in biological tissues. The optical ATP sensor was made by applying two layers of sol-gel coating to the end of an optical fiber probe end. The first layer contained ruthenium complex for sensing changes in ...

  5. An Improved High Yield Total Synthesis and Cytotoxicity Study of the Marine Alkaloid Neoamphimedine: An ATP-Competitive Inhibitor of Topoisomerase IIα and Potent Anticancer Agent

    Directory of Open Access Journals (Sweden)

    Linfeng Li

    2014-09-01

    Full Text Available Recently, we characterized neoamphimedine (neo as an ATP-competitive inhibitor of the ATPase domain of human Topoisomerase IIα. Thus far, neo is the only pyridoacridine with this mechanism of action. One limiting factor in the development of neo as a therapeutic agent has been access to sufficient amounts of material for biological testing. Although there are two reported syntheses of neo, both require 12 steps with low overall yields (≤6%. In this article, we report an improved total synthesis of neo achieved in 10 steps with a 25% overall yield. In addition, we report an expanded cytotoxicity study using a panel of human cancer cell lines, including: breast, colorectal, lung, and leukemia. Neo displays potent cytotoxicity (nM IC50 values in all, with significant potency against colorectal cancer (lowest IC50 = 6 nM. We show that neo is cytotoxic not cytostatic, and that neo exerts cytotoxicity by inducing G2-M cell cycle arrest and apoptosis.

  6. Regulation of dopamine synthesis and release in striatal and prefrontal cortical brain slices

    Energy Technology Data Exchange (ETDEWEB)

    Wolf, M.E.

    1986-01-01

    Brain slices were used to investigate the role of nerve terminal autoreceptors in modulating dopamine (DA) synthesis and release in striatum and prefrontal cortex. Accumulation of dihydroxyphenylalanine (DOPA) was used as an index of tyrosine hydroxylation in vitro. Nomifensine, a DA uptake blocker, inhibited DOPA synthesis in striatal but not prefrontal slices. This effect was reversed by the DA antagonist sulpiride, suggesting it involved activation of DA receptors by elevated synaptic levels of DA. The autoreceptor-selective agonist EMD-23-448 also inhibited striatal but not prefrontal DOPA synthesis. DOPA synthesis was stimulated in both brain regions by elevated K/sup +/, however only striatal synthesis could be further enhanced by sulpiride. DA release was measured by following the efflux of radioactivity from brain slices prelabeled with (/sup 3/H)-DA. EMD-23-448 and apomorphine inhibited, while sulpiride enhanced, the K/sup +/-evoked overflow of radioactivity from both striatal and prefrontal cortical slices. These findings suggest that striatal DA nerve terminals possess autoreceptors which modulate tyrosine hydroxylation as well as autoreceptors which modulate release. Alternatively, one site may be coupled to both functions through distinct transduction mechanisms. In contrast, autoreceptors on prefrontal cortical terminals appear to regulate DA release but not DA synthesis.

  7. Cloning and Experssion of Key Enzyme Gene APT1 for ATP Synthesis in Actinomucor elegans%ATP合成关键酶基因APT1在雅致放射毛霉中的克隆表达

    Institute of Scientific and Technical Information of China (English)

    朱家荣; 杨善岩; 陈丽芬; 杨光辉

    2012-01-01

    为构建遗传稳定的ATP高产工程菌,利用PCR技术扩增酿酒酵母ATP合成关键酶基因APT1,并将其克隆至质粒pCB1004-Pgpd的相应位点,得到有强启动子Pgpd驱动的APT1基因超表达质粒pCB1004-Pgpd-APT1。在PEG-CaCl2介导下,超表达质粒转化雅致放射毛霉原生质体,获得ATP高产工程菌。其ATP产量及摩尔转化率比出发菌株提高44.04%。%To construct the engineering strain with genetic stability which can efficient to synthesize ATP,the key enzyme gene APT1 for ATP synthesis which from Saccharomyces cerevisiae was cloned by PCR.The sequence was cloned to corresponding site of pCB1004-Pgpd.Then the super expression plasmid pCB1004-Pgpd-APT1 which controlled by strong promoter Pgpd was obtained.PEG and CaCl2 mediated protoplast transformation of Actinomucor elegans with super expression plasmid was performed and ATP-overproducing transformants were obtained.The ATP yield and Moore conversion were increased 44.04% in comparison with those from original strain.

  8. Glycolysis and ATP degradation in cod ( Gadus morhua ) at subzero temperatures in relation to thaw rigor

    DEFF Research Database (Denmark)

    Cappeln, Gertrud; Jessen, Flemming

    2001-01-01

    Glycolysis was shown to occur during freezing of cod of decrease in glycogen and an increase in lactate. In addition, the ATP content decreased during freezing. Synthesis of ATP was measured as degradation of glycogen. During storage at -9 and - 12 degreesC it was found that degradation of ATP was...... faster than synthesis of ATP. This was leading to presence of glycogen even at low ATP concentrations. The ATP and glycogen degradation rates and lactate formation rate reached an optimum (both in small samples as well as in whole fish) when stored at -9 degreesC compared to -12 degreesC. Evidence of ATP...

  9. Regulation of prostaglandin E2 synthesis after brain irradiation

    International Nuclear Information System (INIS)

    Purpose: A local tissue reaction, termed neuroinflammation, occurs after irradiation of brain tissue. Previous work suggested that cyclooxygenase (COX)-2 activity was important for changes in gene expression associated with neuroinflammation as well as increased prostaglandin E2 (PGE2) levels seen after radiation treatment. Methods and materials: To begin to determine the contributions of other enzymes involved in PGE2 production, we examined protein levels of COX-1 and COX-2 as well as 2 PGE synthases (membrane and cytosolic PGES) 4 h after 35 Gy single dose irradiation to the brains of C3HeN mice. We also evaluated the effects of specific COX inhibitors on PGE2 production and PGES expression. Results: As expected, COX-2 expression increased after radiation exposure. Brain irradiation also increased tissue protein levels for both PGES isoforms. Specific COX-2 inhibition with NS398 lowered brain PGE2 levels by about 60%. Surprisingly, COX-1 inhibition with SC560 completely prevented the elevation of PGE2 seen after irradiation. Interestingly, NS398 reduced the membrane-associated PGES isoform, whereas SC560 treatment lowered cytosolic isoform levels below those seen in unirradiated controls. Conclusions: Taken together, these data indicate that both cyclooxygenases contribute to PGE2 production in irradiated brain and reveal dependence of PGES isoforms expression on specific cyclooxygenase activities

  10. DNA synthesis and cell division in the adult primate brain

    International Nuclear Information System (INIS)

    It is generally accepted that the adult human brain is incapable of producing new neuron. Even cursory examination of neurologic, neuropathologic, or neurobiological textbooks published during the past 50 years will testify that this belief is deeply entrenched. In his classification of cell populations on the basis of their proliferative behavior, Leblond regarded neurons of the central nervous system as belonging to a category of static, nonrenewing epithelial tissue incapable of expanding or replenishing itself. This belief, however needs to re reexamined for two major reasons: First, as reviewed below, a number of reports have provided evidence of neurogenesis in adult brain of several vertebrate species. Second, the capacity for neurogenesis in the adult primate central nervous system has never been examined by modern methods. In this article the author described recent results from an extensive autoradiographic analysis performed on twelve rhesus monkeys injected with the specific DNA precursor [3H] thymidine at ages ranging from 6 postnatal months to 17 years

  11. MR to CT Registration of Brains using Image Synthesis

    OpenAIRE

    Roy, Snehashis; Carass, Aaron; Jog, Amod; Prince, Jerry L.; Lee, Junghoon

    2014-01-01

    Computed tomography (CT) is the standard imaging modality for patient dose calculation for radiation therapy. Magnetic resonance (MR) imaging (MRI) is used along with CT to identify brain structures due to its superior soft tissue contrast. Registration of MR and CT is necessary for accurate delineation of the tumor and other structures, and is critical in radiotherapy planning. Mutual information (MI) or its variants are typically used as a similarity metric to register MRI to CT. However, u...

  12. Brain-Machine Interfaces for Real-time Speech Synthesis

    OpenAIRE

    Guenther, Frank H.; Jonathan S Brumberg

    2011-01-01

    This paper reports on studies involving brain-machine interfaces (BMIs) that provide near-instantaneous audio feedback from a speech synthesizer to the BMI user. In one study, neural signals recorded by an intracranial electrode implanted in a speech-related region of the left precentral gyrus of a human volunteer suffering from locked-in syndrome were transmitted wirelessly across the scalp and used to drive a formant synthesizer, allowing the user to produce vowels. In a second, pilot study...

  13. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    OpenAIRE

    Igamberdiev, Abir U.; Kleczkowski, Leszek A.

    2015-01-01

    The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, ...

  14. Distinct Effects of Estrogen on Mouse Maternal Behavior: The Contribution of Estrogen Synthesis in the Brain.

    Science.gov (United States)

    Murakami, Gen

    2016-01-01

    Estrogen surge following progesterone withdrawal at parturition plays an important role in initiating maternal behavior in various rodent species. Systemic estrogen treatment shortens the latency to onset of maternal behavior in nulliparous female rats that have not experienced parturition. In contrast, nulliparous laboratory mice show rapid onset of maternal behavior without estrogen treatment, and the role of estrogen still remains unclear. Here the effect of systemic estrogen treatment (for 2 h, 1 day, 3 days, and 7 days) after progesterone withdrawal was examined on maternal behavior of C57BL/6 mice. This estrogen regimen led to different effects on nursing, pup retrieval, and nest building behaviors. Latency to nursing was shortened by estrogen treatment within 2 h. Moreover, pup retrieval and nest building were decreased. mRNA expression was also investigated for estrogen receptor α (ERα) and for genes involved in regulating maternal behavior, specifically, the oxytocin receptor (OTR) and vasopressin receptor in the medial amygdala (MeA) and medial preoptic area (MPOA). Estrogen treatment led to decreased ERα mRNA in both regions. Although OTR mRNA was increased in the MeA, OTR and vasopressin receptor mRNA were reduced in the MPOA, showing region-dependent transcription regulation. To determine the mechanisms for the actions of estrogen treatment, the contribution of estrogen synthesis in the brain was examined. Blockade of estrogen synthesis in the brain by systemic letrozole treatment in ovariectomized mice interfered with pup retrieval and nest building but not nursing behavior, indicating different contributions of estrogen synthesis to maternal behavior. Furthermore, letrozole treatment led to an increase in ERα mRNA in the MeA but not in the MPOA, suggesting that involvement of estrogen synthesis is brain region dependent. Altogether, these results suggest that region-dependent estrogen synthesis leads to differential transcriptional activation due

  15. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

    Full Text Available The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs.

  16. Blocking leukotriene synthesis attenuates the pathophysiology of traumatic brain injury and associated cognitive deficits.

    Science.gov (United States)

    Corser-Jensen, Chelsea E; Goodell, Dayton J; Freund, Ronald K; Serbedzija, Predrag; Murphy, Robert C; Farias, Santiago E; Dell'Acqua, Mark L; Frey, Lauren C; Serkova, Natalie; Heidenreich, Kim A

    2014-06-01

    Neuroinflammation is a component of secondary injury following traumatic brain injury (TBI) that can persist beyond the acute phase. Leukotrienes are potent, pro-inflammatory lipid mediators generated from membrane phospholipids. In the absence of injury, leukotrienes are undetectable in the brain, but after trauma they are rapidly synthesized by a transcellular event involving infiltrating neutrophils and endogenous brain cells. Here, we investigate the efficacy of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP), in blocking leukotriene synthesis, secondary brain damage, synaptic dysfunction, and cognitive impairments after TBI. Male Sprague Dawley rats (9-11weeks) received either MK-886 or vehicle after they were subjected to unilateral moderate fluid percussion injury (FPI) to assess the potential clinical use of FLAP inhibitors for TBI. MK-886 was also administered before FPI to determine the preventative potential of FLAP inhibitors. MK-886 given before or after injury significantly blocked the production of leukotrienes, measured by reverse-phase liquid chromatography coupled to tandem mass spectrometry (RP LC-MS/MS), and brain edema, measured by T2-weighted magnetic resonance imaging (MRI). MK-886 significantly attenuated blood-brain barrier disruption in the CA1 hippocampal region and deficits in long-term potentiation (LTP) at CA1 hippocampal synapses. The prevention of FPI-induced synaptic dysfunction by MK-886 was accompanied by fewer deficits in post-injury spatial learning and memory performance in the radial arm water maze (RAWM). These results indicate that leukotrienes contribute significantly to secondary brain injury and subsequent cognitive deficits. FLAP inhibitors represent a novel anti-inflammatory approach for treating human TBI that is feasible for both intervention and prevention of brain injury and neurologic deficits. PMID:24681156

  17. Acute treatment with fluvoxamine elevates rat brain serotonin synthesis in some terminal regions: An autoradiographic study

    International Nuclear Information System (INIS)

    Introduction: A considerable body of evidence indicates the involvement of the neurotransmitter serotonin (5-HT) in the pathogenesis and treatment of depression. Methods: The acute effect of fluvoxamine, on 5-HT synthesis rates was investigated in rat brain regions, using α-14C-methyl-L-tryptophan as a tracer. Fluvoxamine (25 mg/kg) and saline (control) were injected intraperitoneally, one hour before the injection of the tracer (30 μCi). Results: There was no significant effect of fluvoxamine on plasma free tryptophan. After Benjamini–Hochberg False Discovery Rate correction, a significant decrease in the 5-HT synthesis rate in the fluvoxamine treated rats, was found in the raphe magnus (− 32%), but not in the median (− 14%) and dorsal (− 3%) raphe nuclei. In the regions with serotonergic axon terminals, significant increases in synthesis rates were observed in the dorsal (+ 41%) and ventral (+ 43%) hippocampus, visual (+ 38%), auditory (+ 65%) and parietal (+ 37%) cortex, and the substantia nigra pars compacta (+ 56%). There were no significant changes in the 5-HT synthesis rates in the median (+ 11%) and lateral (+ 24%) part of the caudate-putamen, nucleus accumbens (+ 5%), VTA (+ 16%) or frontal cortex (+ 6%). Conclusions: The data show that the acute administration of fluvoxamine affects 5-HT synthesis rates in a regionally specific pattern, with a general elevation of the synthesis in the terminal regions and a reduction in some cell body structures. The reasons for the regional specific effect of fluvoxamine on 5-HT synthesis are unclear, but may be mediated by the presynaptic serotonergic autoreceptors.

  18. Brain

    Science.gov (United States)

    ... will return after updating. Resources Archived Modules Updates Brain Cerebrum The cerebrum is the part of the ... the outside of the brain and spinal cord. Brain Stem The brain stem is the part of ...

  19. The corticosterone synthesis inhibitor metyrapone decreases dopamine D1 receptors in the rat brain

    International Nuclear Information System (INIS)

    Experiments were performed to examine the effect of metyrapone, an inhibitor of corticosterone synthesis, on the level of dopamine D1 receptors and their transcripts in the caudate-putamen, nucleus accumbens and olfactory tubercle of the rat brain. The binding to dopamine D1 receptors was measured by receptor autoradiography using the specific D1 receptor antagonist [3H]SCH 23390. The level of dopamine D1 receptor messenger RNA was determined by in situ hybridization histochemistry. The results obtained have shown that metyrapone (two injections of 150 and 50 mg/kg, i.p., given 20 and 3 h before killing, respectively) induced a decrease in the D1 receptor-specific binding in the studied areas of the rat brain. In the caudate-putamen, the decrease in [3H]SCH 23390 binding was stronger in the medial (31-39%) than in the lateral part (24-27%). Decreases similar to those in the caudate-putamen were observed in the nucleus accumbens (21%) and olfactory tubercle (32%). Furthermore, metyrapone decreased the level of dopamine D1 receptor messenger RNA in the caudate-putamen (17-28%), nucleus accumbens (20%) and olfactory tubercle (18%).In conclusion, our study indicates that glucocorticoids might be involved in the regulation of dopamine D1 receptor level in the rat brain, since metyrapone (which inhibits the synthesis of these hormones) decreases the messenger RNA encoding D1 receptor synthesis, as well as the specific binding to this receptor. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  20. Inhibition of cerebroside synthesis in the brains of mice treated with L-cycloserine

    International Nuclear Information System (INIS)

    Subcutaneous injection of L-cycloserine resulted in a 28% reduction in cerebroside levels in mouse brain but had no effect on the levels of gangliosides. In contrast, intraperitoneal injection results in a reduction of ganglioside as well as cerebroside + sulfatide levels. The route of injection influenced the degree of 3-ketodihydrosphingosine synthase inhibition. Intraperitoneal injection caused a rapid decrease in synthase activity followed by recovery over 48 hr, whereas subcutaneous injection resulted in no inhibition over this time; only after daily injection for a week was synthase activity reduced 35%. One week following cessation of L-cycloserine administration, enzyme activity had recovered, whereas the cerebroside level continued to fall. All lipids and enzymes showed normal levels 3 weeks post-cycloserine administration. L-[3H]serine incorporation into glycolipids showed that cerebroside synthesis was most affected, whereas sulfatide synthesis was less affected. One week after cessation of cycloserine treatment, cerebroside synthesis was still severely inhibited, whereas sulfatide levels were near normal. Two weeks after cessation of L-cycloserine administration, synthesis of these glycolipids was similar to that of controls

  1. Metabolomic Analysis of Differential Changes in Metabolites during ATP Oscillations in Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Hyuck Joon Kwon

    2013-01-01

    Full Text Available Prechondrogenic condensation is a critical step for skeletal pattern formation. Recent studies reported that ATP oscillations play an essential role in prechondrogenic condensation. However, the molecular mechanism to underlie ATP oscillations remains poorly understood. In the present study, it was investigated how changes in metabolites are implicated in ATP oscillations during chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS. CE-TOF-MS detected 93 cationic and 109 anionic compounds derived from known metabolic pathways. 15 cationic and 18 anionic compounds revealed significant change between peak and trough of ATP oscillations. These results implicate that glycolysis, mitochondrial respiration and uronic acid pathway oscillate in phase with ATP oscillations, while PPRP and nucleotides synthesis pathways oscillate in antiphase with ATP oscillations. This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate in antiphase with the ATP-consuming PPRP/nucleotide synthesis pathway during chondrogenesis.

  2. Role of glycogenolysis in memory and learning: regulation by noradrenaline, serotonin and ATP

    Directory of Open Access Journals (Sweden)

    Marie Elizabeth Gibbs

    2016-01-01

    Full Text Available This paper reviews the role played by glycogen breakdown (glycogenolysis and glycogen re-synthesis in memory processing in two different chick brain regions, (1 the hippocampus and (2 the avian equivalent of the mammalian cortex, the intermediate medial mesopallium (IMM. Memory processing is regulated by the neuromodulators noradrenaline and serotonin soon after training and glycogen breakdown and re-synthesis are involved. In day-old domestic chicks, memory formation is dependent on the breakdown of glycogen (glycogenolysis at three specific times during the first 60 min after learning (around 2.5, 30 and 55 min. The chicks learn to discriminate in a single trial between beads of two colours and tastes. Inhibition of glycogen breakdown by the inhibitor of glycogen phosphorylase 1,4-dideoxy-1,4-imino-D-arabinitol (DAB given at specific times prior to the formation of long-term memory prevents memory forming. Noradrenergic stimulation of cultured chicken astrocytes by a selective β2-adrenergic (AR agonist reduces glycogen levels and we believe that in vivo this triggers memory consolidation at the second stage of glycogenolysis. Serotonin acting at 5-HT2B receptors acts on the first stage, but not on the second. We have shown that noradrenaline, acting via post-synaptic α2-ARs, is also responsible for the synthesis of glycogen and our experiments suggest that there is a readily accessible labile pool of glycogen in astrocytes which is depleted within 10 min if glycogen synthesis is inhibited. Endogenous ATP promotion of memory consolidation at 2.5 and 30 min is also dependent on glycogen breakdown. ATP acts at P2Y1 receptors and the action of thrombin suggests that it causes the release of internal calcium ([Ca2+]i] in astrocytes. Glutamate and GABA, the primary neurotransmitters in the brain, cannot be synthesized in neurons de novo. Neurons rely on astrocytic glutamate synthesis, requiring glycogenolysis.

  3. ATP generation in Leishmania donovani amastigote form

    Directory of Open Access Journals (Sweden)

    Anup Kumar Roy

    2013-06-01

    Full Text Available Leishmania is the causative agent of various forms of leishmaniasis, a significant cause of morbidity and mortality. The clinical manifestations of the disease range from selfhealing cutaneous and mucocutaneous skin ulcers to a fatal visceral form named visceral leishmaniasis or kala-azar. The differentiation of Leishmania parasites from the insect stage, the promastigote, towards the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect to mammal transmission. The survival of amastigote stage is dependent on that of the host. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. The amastigote form of L. donovani is independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by 20-fold excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial FoF1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation, whereas NADHfumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. We conclude that substrate level phosphorylation is essential for the growth of L. donovani amastigotes.

  4. Synthesis and characterization of brain penetrant prodrug of neuroprotective D-264: Potential therapeutic application in the treatment of Parkinson's disease.

    Science.gov (United States)

    Dholkawala, Fahd; Voshavar, Chandrashekhar; Dutta, Aloke K

    2016-06-01

    Parkinson's disease (PD) is one of the major debilitating neurodegenerative disorders affecting millions of people worldwide. Progressive loss of dopamine neurons resulting in development of motor dysfunction and other related non-motor symptoms is the hallmark of PD. Previously, we have reported on the neuroprotective property of a potent D3 preferring agonist D-264. In our goal to increase the bioavailability of D-264 in the brain, we have synthesized a modified cysteine based prodrug of D-264 and evaluated its potential in crossing the blood-brain barrier. Herein, we report the synthesis of a novel modified cysteine conjugated prodrug of potent neuroprotective D3 preferring agonist D-264 and systematic evaluation of the hydrolysis pattern of the prodrug to yield D-264 at different time intervals in rat plasma and brain homogenates using HPLC analysis. Furthermore, we have also performed in vivo experiments with the prodrug to evaluate its enhanced brain penetration ability. PMID:26994936

  5. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    Science.gov (United States)

    Brand, M D; Lehninger, A L

    1977-05-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  6. X-ray dose-effect relationship on unscheduled DNA synthesis and spontaneous unscheduled DNA synthesis in mouse brain cells studied in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Korr, H.; Koeser, K.; Oldenkott, S.; Schmidt, H.; Schultze, B.

    1989-01-01

    X-irradiation of the head of adult mice leads to DNA repair synthesis (unscheduled DNA synthesis, UDS) in non-proliferating cells of the brain as shown autoradiographically after injection of /sup 3/H-thymidine and subsequent irradiation. The extent of UDS induced by one and the same X-ray dose varies between different cell types and also between different brain areas. Within the range of X-ray doses studied (2 to 100 Gy) a linear dose effect relationship was observed. No evidence of a saturation effect was found. The slopes of the regression lines for the dose effect relationship differ considerably for the different cell types. Two interesting correlations were found, if the present results were compared with other data in the literature: (i) There seems to be a correlation between the extent of UDS and radiosensitivity of different cell types, the cells with the low DNA repair synthetic rates being more radiosensitive. (ii) The extent of UDS of the different cell types correlates well with the extent of protein synthesis of the corresponding cell types. Apart from radiation induced UDS, spontaneous UDS was found to occur in sham-irradiated animals. The extent of spontaneous UDS also differs considerably between different cell types as well as between different brain areas. The increase of spontaneous UDS with increasing duration of immobilization of the animals during sham irradiation suggests a relationship between spontaneous UDS and stress.

  7. Brain protein synthesis in normal and demented patients. A study by P.E.T. with 11C-L methionine

    International Nuclear Information System (INIS)

    A compartmental model representing protein synthesis in the brain was validated experimentally in 9 baboons. After sequential injections of 11C, 3H and 14C methionines on the same animal, followed by P.E.T. recording of the γ activity in a chosen brain section with time, the distribution of methionine injected into the different compartments of the model after a bolus was measured by crushing and precipitation with T.C.A. The agreement between direct in vitro findings and computed results is excellent. This method of studying brain protein synthesis in vivo was applied to 28 Alzheimer dementia cases and 20 normal subjects of the same age. The correlation between the results of clinical and psychometric tests and the brain protein synthesis activity confirms an anomaly in this biochemical synthesis process during the illness. A 65% fall in activity may be found in the frontal lobes of certain patients

  8. Adenosine and ATP Receptors in the Brain

    Czech Academy of Sciences Publication Activity Database

    Burnstock, G.; Fredholm, B. B.; Verkhratsky, Alexei

    2011-01-01

    Roč. 11, č. 8 (2011), s. 973-1011. ISSN 1568-0266 R&D Projects: GA ČR GA309/08/1381; GA ČR GA305/08/1384 Institutional research plan: CEZ:AV0Z50390703 Keywords : Ischaemia * CNS * glia Subject RIV: FH - Neurology Impact factor: 4.174, year: 2011

  9. Ectopic ATP synthase in endothelial cells: a novel cardiovascular therapeutic target.

    Science.gov (United States)

    Fu, Yi; Zhu, Yi

    2010-01-01

    Adenosine triphosphate (ATP) synthase produces ATP in cells and is found on the inner membrane of mitochondria or the cell plasma membrane (ectopic ATP synthase). Here, we summarize the functions of ectopic ATP synthase in vascular endothelial cells (ECs). Ectopic ATP synthase is involved in adenosine metabolism on the cell surface through its ATP generation or hydrolysis activity. The ATP/ADP generated by the enzyme on the plasma membrane can bind to P2X/P2Y receptors and activate the related signalling pathways to regulate endothelial function. The β-chain of ectopic ATP synthase on the EC surface can recruit inflammatory cells and activate cytotoxic activity to damage ECs and induce vascular inflammation. Angiostatin and other angiogenesis inhibitors can have anti-angiogenic functions by inhibiting ectopic ATP synthase on ECs. Moreover, ectopic ATP synthase on ECs is a receptor for apoA-I, the acceptor of cholesterol efflux, which implies that endothelial ectopic ATP synthase is involved in cholesterol metabolism. Coupling factor 6 (CF6), a part of ectopic ATP synthase, is released from ECs and can inhibit prostacyclin synthesis and promote nitric oxide (NO) degradation to enhance NO bioactivity. Because ATP/ADP generated by ectopic ATP synthase can induce NO production, substances such as CF6 can inhibit NO generation by inhibiting surface ATP/ADP production. Thus, the components of ectopic ATP synthase are associated with regulation of vascular tone. Through these functions, ectopic ATP synthase on ECs is considered a potential and novel therapeutic target for atherosclerosis, hypertension and lipid disorders. PMID:21247400

  10. Phylogenetic Analysis of the Thylakoid ATP/ADP Carrier Reveals New Insights into Its Function Restricted to Green Plants

    OpenAIRE

    Spetea, Cornelia; Pfeil, Bernard E.; Schoefs, Benoît

    2012-01-01

    ATP is the common energy currency of cellular metabolism in all living organisms. Most of them synthesize ATP in the cytosol or on the mitochondrial inner membrane, whereas land plants, algae, and cyanobacteria also produce it on the thylakoid membrane during the light-dependent reactions of photosynthesis. From the site of synthesis, ATP is transported to the site of utilization via intracellular membrane transporters. One major type of ATP transporters is represented by the mitochondrial AD...

  11. Phylogenetic analysis of the thylakoid ATP/ADP carrier reveals new insights into its function restricted to green plants

    OpenAIRE

    Cornelia eSpetea; Pfeil, Bernard E.; Benoit eSchoefs

    2012-01-01

    ATP is the common energy currency of cellular metabolism in all living organisms. Most of them synthesize ATP in the cytosol or on the mitochondrial inner membrane, whereas land plants, algae and cyanobacteria also produce it on the thylakoid membrane during the light-dependent reactions of photosynthesis. From the site of synthesis, ATP is transported to the site of utilization via intracellular membranes transporters. One major type of ATP transporter is represented by the mitochondrial ADP...

  12. F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

    OpenAIRE

    Rak, Malgorzata; Tzagoloff, Alexander

    2009-01-01

    The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the b...

  13. Arginase and Arginine Decarboxylase - Where Do the Putative Gate Keepers of Polyamine Synthesis Reside in Rat Brain?

    Directory of Open Access Journals (Sweden)

    Daniela Peters

    Full Text Available Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine

  14. ATP5A1 and ATP5B are highly expressed in glioblastoma tumor cells and endothelial cells of microvascular proliferation.

    Science.gov (United States)

    Xu, Guiyan; Li, Jian Yi

    2016-02-01

    Glioblastoma (GBM) is the most common primary malignant brain tumor. Microvascular proliferation is one of the characteristic pathologic features of GBM. Mitochondrial dysfunction plays an important role in the pathogenesis of GBM. In this study, microvascular proliferation from GBM and normal brain blood vessels were laser microdissected and total RNA was isolated from these microvasculatures. The difference of mRNA expression profiles among GBM microvasculature, normal brain blood vessels and GBM tumor cells was evaluated by mitochondria and metabolism PCR gene arrays. It was found that the mRNA levels of ATP5A1 and ATP5B in GBM tumor cells as well as microvascular proliferation were significantly higher compared with normal brain blood vessels. Immunohistochemical stains with anti-ATP5A1 antibody or anti-ATP5B antibody were performed on tissue microarray, which demonstrated strongly positive expression of ATP5A1 and ATP5B in GBM tumor cells and GBM microvascular proliferation while normal blood vessels were negative. By analyzing The Cancer Genome Atlas data sets for GBM and other cancers, genomic DNA alterations (mutation, amplification or deletion) were less likely the reason for the high expression of ATP5A1 and ATP5B in GBM. Our miRNA microarray data showed that miRNAs that target ATP5A1 or ATP5B were down-regulated, which might be the most likely reason for the high expression of ATP5A1 and ATP5B in GBM tumor cells and microvascular proliferation. These findings help us better understand the pathogenesis of GBM, and agents against ATP5A1 and/or ATP5B might effectively kill both tumor cells and microvascular proliferation in GBM. MiRNAs, such as Let-7f, miR-16, miR-23, miR-100 and miR-101, that target ATP5A1 or ATP5B, might be potential therapeutic agents for GBM. PMID:26526033

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

    Directory of Open Access Journals (Sweden)

    Ip Virginia

    2010-09-01

    Full Text Available Abstract 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

  16. Regulation of ongoing DNA synthesis in normal and neoplastic brain tissue

    OpenAIRE

    Yakisich, Juan Sebastián

    2005-01-01

    The treatment of human brain tumour is challenging in part due to the blood brain barrier and in part due to the specific biology of brain tumours that confer resistance to chemotherapy. For instance, the 5 years survival rate for patients carrying intracranial glioblastoma multiforme has remained at 4-5 % for the last 30 years. The knowledge of the brain tumour biology as well as the biology of the normal brain tissue would help to design new therapeutic strategies and to d...

  17. Autism Post-Mortem Neuroinformatic Resource: The Autism Tissue Program (ATP) Informatics Portal

    Science.gov (United States)

    Brimacombe, Michael B.; Pickett, Richard; Pickett, Jane

    2007-01-01

    The Autism Tissue Program (ATP) was established to oversee and manage brain donations related to neurological research in autism. The ATP Informatics Portal (www.atpportal.org) is an integrated data access system based on Oracle technology, developed to provide access for researchers to information on this rare tissue resource. It also permits…

  18. Double-lock ratchet mechanism revealing the role of  SER-344 in FoF1 ATP synthase

    KAUST Repository

    Beke-Somfai, T.

    2011-03-07

    In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi→ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γP-OADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

  19. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  20. Modulation of Nucleotide Specificity of Thermophilic FoF1-ATP Synthase by ϵ-Subunit

    OpenAIRE

    Suzuki, Toshiharu; Wakabayashi, Chiaki; TANAKA, Kazumi; Feniouk, Boris A.; Yoshida, Masasuke

    2011-01-01

    The C-terminal two α-helices of the ϵ-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm (“up-state”) and a retracted hairpin (“down-state”). As ATP becomes poor, ϵ changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ϵ in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the up-state ϵ was achi...

  1. The CADE ATP System Competition — CASC

    OpenAIRE

    Sutcliffe, Geoff; University of Miami.

    2016-01-01

    The CADE ATP System Competition (CASC) is an annual evaluation of fully automatic automated theorem proving (ATP) systems for classical logic — the world championship for such systems. CASC provides a public evaluation of the relative capabilities of ATP systems, and aims stimulate ATP research towards the development of more powerful ATP systems. Over the years CASC has been a catalyst for impressive improvements in ATP.

  2. An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.

    Science.gov (United States)

    Baricordi, O R; Ferrari, D; Melchiorri, L; Chiozzi, P; Hanau, S; Chiari, E; Rubini, M; Di Virgilio, F

    1996-01-15

    We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation. PMID:8555491

  3. Synthesis and evaluation of [/sup 125/I]iodothienoperidol as a potential receptor site directed brain imaging agent

    International Nuclear Information System (INIS)

    This study was undertaken to design and evaluate radioligands for the noninvasive quantification of dopamine receptors in the brain. The approach involved the preparation of the iodothienyl analog I of haloperidol II, a well characterized dopamine antagonist which has been labeled with F-18 and C-11. The synthesis involved the addition of 5-trimethylstannyl-2-thienyllithium so the piperidone intermediate. The product was characterized by spectroscopic and analytic methods and radioiodinated via electrophilic iododestannylation to yield the product in 75-85% isolated yield. The tissue distribution of the radiochemical was evaluated in rats as a function of time, 0.25-2 hrs, and in the presence or absence of haloperidol (1 mg/kg) to measure receptor binding. The results indicated that the 0.25 h uptake in the brain was high (2.2% ID) and that the washout of the activity was relatively slow, 1.3% ID present at 2 hr. The Br/B1 values remained relatively constant over that time interval (9.3-12.1:1). Coadministration of 1 mg/kg haloperidol markedly reduced the uptake in the brain at 0.25 and 2 hr (55% and 62%) with much less of an effect on the nontarget tissues. The study indicates that the authors have prepared a radiotracer, labeled with iodine, that demonstrates both good brain uptake and selectivity as well as a specific binding site component

  4. Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase

    Science.gov (United States)

    Sellem, Carole H.; di Rago, Jean-Paul; Lasserre, Jean-Paul; Ackerman, Sharon H.; Sainsard-Chanet, Annie

    2016-01-01

    Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8–15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before. PMID:27442014

  5. Small amounts of functional ATP7A protein permit mild phenotype

    DEFF Research Database (Denmark)

    Møller, Lisbeth Birk

    2015-01-01

    . The location of missense mutations on structural models of the ATP7A protein suggests that affected conserved residues generally lead to a severe phenotype. The ATP7A protein traffics within the cells. At low copper levels, ATP7A locates to the Trans-Golgi Network (TGN) to load cuproenzymes with...... copper, whereas at higher concentrations, ATP7A shifts to the post-Golgi compartments or to the plasma membrane to export copper out of the cell. Impaired copper-regulation trafficking has been observed for ATP7A mutants, but its impact on the clinical outcome is not clear. The major problem in patients...... with MD seems to be insufficient amounts of copper in the brain. In fact, prenatal treatment of mottled mice as a model for human MD with a combination of chelator and copper, produces a slight increase in copper levels in the brain which perhaps leads to longer survival and more active behavior. In...

  6. Modeling the effects of hypoxia on ATP turnover in exercising muscle

    Science.gov (United States)

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

    1992-01-01

    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.

  7. Effect of Granule of Whangee Clearing Mind on IL-6, Na +-K +-ATP Enzyme and Brain Moisture Capacity in Rats with Brain Hemorrhage Belonging to Phlegm-heat and FU-organ Sthenia Syndrome%黄竹清脑颗粒对痰热腑实证脑出血大鼠IL-6、Na+-K+-ATP酶活性及脑含水量的影响

    Institute of Scientific and Technical Information of China (English)

    刘淑霞; 白雅; 任惠锋; 石春明

    2009-01-01

    目的:探讨黄竹清脑颗粒对痰热腑实型脑出血大鼠模型血清中IL-6、Na+-K+-ATP酶的活性及脑含水量的影响.方法:采用脑内注射胶原酶Ⅰ-肝素混合液配合大鼠自体粪便灌胃的方法建立大鼠脑出血痰热腑实证模型,将SD大鼠随机分为空白对照组、黄竹清脑颗粒大、中、小剂量组、二陈汤提取液组、鲜竹沥口服液组、痰热腑实证模型组,分别采用放免法及光化学比色法测定各组大鼠血清中IL-6、脑组织匀浆中Na+-K+-ATP酶的活性及脑组织含水量的变化.结果:(1)模型组与空白组相比,血清中IL-6水平及脑组织含水量明显升高(P<0.01),脑组织中Na+-K+-ATP酶明显降低(P<0.01);(2)黄竹清脑颗粒大、中剂量组疗效均明显优于二陈汤组、鲜竹沥组及黄竹清脑颗粒小剂量组.结论:黄竹清脑颗粒可显著提高脑组织中Na+-K+-ATP酶的活性,降低血清中IL-6的含量,具有减轻细胞内水肿、抑制炎性反应,保护脑神经细胞的作用;并对痰热腑实型脑出血有明显的改善作用.%Objective: To investigate the effect of Granule of Whangee Clearing Mind on IL-6, Na + -K + -ATP enzyme and brain moisture capacity in rats with brain hemorrhage belonging to phlegm-heat and FU-organ sthenia syndrome. Method : The model of phlegm-heat and FU-organ sthenia syndrome was made by injecting mixed liquor of clostridiopeptidase Ⅰ-calparine and autos dejecta to brain. SD rats were divided into blank control group, large dose group of Granule of Whangee Clearing Mind, middle close group group of Granule of Whangee Clearing Mind, low dose group of Granule of Whangee Clearing Mind, Er-chen Decoction extract group, bamboo juice group and model group. Determine IL-6 in serum, Na + -K + -ATP enzymatic activity in brain homogenate and brain moisture model group increased more obviously than in blank control group ( P < 0.01 ), and Na + -K + -ATP enzyme de- Whangee Clearing Mind were better remarkably

  8. Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK).

    Science.gov (United States)

    Kleszczyński, Konrad; Zillikens, Detlef; Fischer, Tobias W

    2016-09-01

    Melatonin is an ubiquitous molecule with a variety of functions including potent antioxidative properties. Due to its lipophilic character, it easily crosses cellular and intracellular membranes and reaches all subcellular organelles. Because of its ability to scavenge free radicals, melatonin protects against oxidative stress, for example, induced by ultraviolet radiation (UVR). Here, we investigated, in a dose-dependent (0, 10, 25, and 50 mJ/cm(2) ) and time-dependent (0, 4, 24, 48 hr post-UVR) manner, whether melatonin prevents the UVR-mediated alterations in ATP synthesis and the generation of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEK). Additionally, we evaluated the molecular mechanism of action of melatonin with regard to activation of phase-2 antioxidative enzymes via nuclear erythroid 2-related factor (Nrf2). We found that (i) melatonin counteracted UVR-induced alterations in the ATP synthesis and reduced free radical formation; (ii) melatonin induced the translocation of Nrf2 transcription factor from the cytosol into the nucleus resulting in, (iii) melatonin enhanced gene expression of phase-2 antioxidative enzymes including γ-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1), and NADPH: quinone dehydrogenase-1 (NQO1) representing an elevated antioxidative response of keratinocytes. These results suggest that melatonin not only directly scavenges ROS, but also significantly induces the activation of phase-2 antioxidative enzymes via the Nrf2 pathway uncovering a new action mechanism that supports the ability of keratinocytes to protect themselves from UVR-mediated oxidative stress. PMID:27117941

  9. Stepwise Assembly of Dimeric F1Fo-ATP Synthase in Mitochondria Involves the Small Fo-Subunits k and i

    OpenAIRE

    Wagner, Karina; Perschil, Inge; Fichter, Christiane D.; van der Laan, Martin

    2010-01-01

    F1Fo-ATP synthase is a key enzyme of oxidative phosphorylation that is localized in the inner membrane of mitochondria. It uses the energy stored in the proton gradient across the inner mitochondrial membrane to catalyze the synthesis of ATP from ADP and phosphate. Dimeric and higher oligomeric forms of ATP synthase have been observed in mitochondria from various organisms. Oligomerization of ATP synthase is critical for the morphology of the inner mitochondrial membrane because it supports t...

  10. Synthesis of a novel 99mTc nitrido radiopharmaceutical with isopropyl xanthate, showing brain uptake

    International Nuclear Information System (INIS)

    In the present study, isopropyl xanthate (IPXT) ligand was labeled with the [99mTc≡N]2+ core successfully to obtain the 99mTcN(IPXT)2 complex with high radiochemical purity. No decomposition of the complex at room temperature was observed over a period of 6 h. Its partition coefficient indicated that it was a good lipophilic complex. The electrophoresis results showed that the complex was neutral. Biodistribution in mice demonstrated that the complex accumulated in the brain with high uptake and good retention. The brain uptake (ID%/g) was 1.95, 1.58 and 1.86 at 5-, 30- and 60-min post-injection, respectively. As compared with other reported 99mTcN-xanthates, the 99mTcN(IPXT)2 complex showed higher brain uptake and better brain retention, suggesting potential usefulness of the complex as a brain perfusion imaging agent

  11. Brain white matter development is associated with a human-specific haplotype increasing the synthesis of long chain fatty acids.

    Science.gov (United States)

    Peters, Bart D; Voineskos, Aristotle N; Szeszko, Philip R; Lett, Tristram A; DeRosse, Pamela; Guha, Saurav; Karlsgodt, Katherine H; Ikuta, Toshikazu; Felsky, Daniel; John, Majnu; Rotenberg, David J; Kennedy, James L; Lencz, Todd; Malhotra, Anil K

    2014-04-30

    The genetic and molecular pathways driving human brain white matter (WM) development are only beginning to be discovered. Long chain polyunsaturated fatty acids (LC-PUFAs) have been implicated in myelination in animal models and humans. The biosynthesis of LC-PUFAs is regulated by the fatty acid desaturase (FADS) genes, of which a human-specific haplotype is strongly associated with ω-3 and ω-6 LC-PUFA concentrations in blood. To investigate the relationship between LC-PUFA synthesis and human brain WM development, we examined whether this FADS haplotype is associated with age-related WM differences across the life span in healthy individuals 9-86 years of age (n = 207). Diffusion tensor imaging was performed to measure fractional anisotropy (FA), a putative measure of myelination, of the cerebral WM tracts. FADS haplotype status was determined with a single nucleotide polymorphism (rs174583) that tags this haplotype. Overall, normal age-related WM differences were observed, including higher FA values in early adulthood compared with childhood, followed by lower FA values across older age ranges. However, individuals homozygous for the minor allele (associated with lower LC-PUFA concentrations) did not display these normal age-related WM differences (significant age × genotype interactions, p(corrected) < 0.05). These findings suggest that LC-PUFAs are involved in human brain WM development from childhood into adulthood. This haplotype and LC-PUFAs may play a role in myelin-related disorders of neurodevelopmental origin. PMID:24790207

  12. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    OpenAIRE

    Villamor, J. G.; Kaschani, F.; Colby, T; Oeljeklaus, J.; Zhao, D; Kaiser, M.; Patricelli, M. P.; R. A. L. van der Hoorn

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding prot...

  13. Does ATP cross the cell plasma membrane.

    OpenAIRE

    Chaudry, I. H.

    1982-01-01

    Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes.

  14. Brain-inspired cheminformatics of drug-target brain interactome, synthesis, and assay of TVP1022 derivatives.

    Science.gov (United States)

    Romero-Durán, Francisco J; Alonso, Nerea; Yañez, Matilde; Caamaño, Olga; García-Mera, Xerardo; González-Díaz, Humberto

    2016-04-01

    The use of Cheminformatics tools is gaining importance in the field of translational research from Medicinal Chemistry to Neuropharmacology. In particular, we need it for the analysis of chemical information on large datasets of bioactive compounds. These compounds form large multi-target complex networks (drug-target interactome network) resulting in a very challenging data analysis problem. Artificial Neural Network (ANN) algorithms may help us predict the interactions of drugs and targets in CNS interactome. In this work, we trained different ANN models able to predict a large number of drug-target interactions. These models predict a dataset of thousands of interactions of central nervous system (CNS) drugs characterized by > 30 different experimental measures in >400 different experimental protocols for >150 molecular and cellular targets present in 11 different organisms (including human). The model was able to classify cases of non-interacting vs. interacting drug-target pairs with satisfactory performance. A second aim focus on two main directions: the synthesis and assay of new derivatives of TVP1022 (S-analogues of rasagiline) and the comparison with other rasagiline derivatives recently reported. Finally, we used the best of our models to predict drug-target interactions for the best new synthesized compound against a large number of CNS protein targets. PMID:26721628

  15. Altered pattern of brain dopamine synthesis in male adolescents with attention deficit hyperactivity disorder

    OpenAIRE

    Waters Nicholas; Waters Susanna; Fernell Elisabeth; Forssberg Hans; Tedroff Joakim

    2006-01-01

    Abstract Background Limited data from positron emission tomography (PET) studies of subjects with attention-deficit/hyperactivity disorder (ADHD) indicate alterations in brain dopamine neurotransmission. However, these studies have used conventional univariate approaches that are less sensitive to detect complex interactions that may exist between different brain dopamine pathways and individual symptoms of ADHD. We aimed to investigate these potential interactions in adolescents with ADHD. M...

  16. Design, synthesis and preliminary biological evaluation of brain targeting L-ascorbic acid prodrugs of ibuprofen

    Institute of Scientific and Technical Information of China (English)

    Xue-Ying Wu; Xiao-Cen Li; Jie Mi; Jing You; Li Hai

    2013-01-01

    L-Ascorbic acid (AA,vitamin C) exhibits a high concentration in the brain.The transportation of AA in brain is mainly mediated by the glucose transporter 1 (GLUT1) and the Na+-dependent vitamin C transporter SVCT2.While L-ascorbic acid C6-O conjugation has been investigated as a tool to enhance brain drug delivery,C5-O conjugation and C5-O & C6-O conjugation as brain targeting tools have not been reported.In this letter,ibuprofen was linked directly to C5-O,C6-O and C5-O & C6-O positions of L-ascorbic acid with eater bonds,providing prodrug 1,2 and 3,respectively,to improve their targeting abilities in the brain.Prodrug 1,2 and 3 were synthesized in facile ways with good yields.And the preliminary evaluation in vivo illustrated that prodrug 2 had a better targeting ability than prodrug 1.Moreover,prodrug 3,whose C5-O & C6-O positions were both modified,had good targeting ability for brain which will provide an important evidence for our further study on C5-O-& C6-O-di-derivatives of L-ascorbic acid.

  17. 31P magnetization transfer measurements of Pi→ATP flux in exercising human muscle

    Science.gov (United States)

    Savage, David B.; Williams, Guy B.; Porter, David; Carpenter, T. Adrian; Brindle, Kevin M.; Kemp, Graham J.

    2016-01-01

    Fundamental criticisms have been made over the use of 31P magnetic resonance spectroscopy (MRS) magnetization transfer estimates of inorganic phosphate (Pi)→ATP flux (VPi-ATP) in human resting skeletal muscle for assessing mitochondrial function. Although the discrepancy in the magnitude of VPi-ATP is now acknowledged, little is known about its metabolic determinants. Here we use a novel protocol to measure VPi-ATP in human exercising muscle for the first time. Steady-state VPi-ATP was measured at rest and over a range of exercise intensities and compared with suprabasal oxidative ATP synthesis rates estimated from the initial rates of postexercise phosphocreatine resynthesis (VATP). We define a surplus Pi→ATP flux as the difference between VPi-ATP and VATP. The coupled reactions catalyzed by the glycolytic enzymes GAPDH and phosphoglycerate kinase (PGK) have been shown to catalyze measurable exchange between ATP and Pi in some systems and have been suggested to be responsible for this surplus flux. Surplus VPi-ATP did not change between rest and exercise, even though the concentrations of Pi and ADP, which are substrates for GAPDH and PGK, respectively, increased as expected. However, involvement of these enzymes is suggested by correlations between absolute and surplus Pi→ATP flux, both at rest and during exercise, and the intensity of the phosphomonoester peak in the 31P NMR spectrum. This peak includes contributions from sugar phosphates in the glycolytic pathway, and changes in its intensity may indicate changes in downstream glycolytic intermediates, including 3-phosphoglycerate, which has been shown to influence the exchange between ATP and Pi catalyzed by GAPDH and PGK. PMID:26744504

  18. The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

    OpenAIRE

    Suzuki, Haruka; Kuroda, Hiroshi; Yukawa, Yasushi; Sugiura, Masahiro

    2011-01-01

    The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the ...

  19. Synthesis of 11C-methylated inulin as a radiopharmaceutical for imaging brain edema and pulmonary edema

    International Nuclear Information System (INIS)

    11C-methylated inulin, supposedly useful for imaging of brain edema and pulmonary edema, was prepared using cyclotron produced 11CO2. The synthesis consists of the production of 11C-methyl iodide and its coupling with inulin alkoxide sodium in dimethylsulfoxide as solvent. 11C labeled inulin was purified by alcohol precipitation. The radiochemical yield of pure 11C-inulin was 34% of 11CO2 30 min after the end of bombardment. The blood clearance and body distribution of 11C was observed in rabbits after i.v. injection of 11C-inulin. The blood clearance curve was composed of a sum of three exponential functions. The gamma camera image showed that the 11C activity in blood moved quickly to kidneys and urine and a small dose of radioactivity remained persistently in edematous tissues, i.e. the edematous lung tissues produced by oleic acid treatment. (orig.)

  20. ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

    Science.gov (United States)

    Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

    2015-12-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering.

  1. Altered pattern of brain dopamine synthesis in male adolescents with attention deficit hyperactivity disorder

    Directory of Open Access Journals (Sweden)

    Waters Nicholas

    2006-12-01

    Full Text Available Abstract Background Limited data from positron emission tomography (PET studies of subjects with attention-deficit/hyperactivity disorder (ADHD indicate alterations in brain dopamine neurotransmission. However, these studies have used conventional univariate approaches that are less sensitive to detect complex interactions that may exist between different brain dopamine pathways and individual symptoms of ADHD. We aimed to investigate these potential interactions in adolescents with ADHD. Methods We used a 3D PET scan to measure utilization of native L-[11C]-DOPA to map dopamine presynaptic function in various cortical, striatal and midbrain regions in a group of 8 male adolescents with ADHD and 6 age matched controls. To evaluate the interactions between the studied brain regions, multivariate statistical methods were used. Results Abnormal dopaminergic function was found in multiple brain regions of patients with ADHD. A main finding was lower L-[11C]-DOPA utilization in adolescent with ADHD as compared to control subjects, especially in subcortical regions. This pattern of dopaminergic activity was correlated specifically with symptoms of inattention. Conclusion Dopamine signalling in the brain plays an important modulatory role in a variety of motor and cognitive functions. We have identified region-specific functional abnormalities in dopaminergic function, which may help better account for the symptoms of ADHD.

  2. ATP interaction with the open state of the K(ATP) channel.

    OpenAIRE

    Enkvetchakul, D; Loussouarn, G.; Makhina, E; Nichols, C.G.

    2001-01-01

    The mechanism of ATP-sensitive potassium (K(ATP)) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP2) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact wi...

  3. Synthesis, labeling with 99mTc and biokinetics of brains scintigraphy diaminodithiol perfusion radiopharmaceuticals

    International Nuclear Information System (INIS)

    The recent tomography status using radiopharmaceuticals have been contributing greatly with the 'age of certainty' in the diagnosis examination of syndromes, pathologies and clinical signs, because they can evidence some phenomena occurring in a molecular manner. The purpose of this work have had the development of new diaminodithiol (DADT) perfusion radiopharmaceuticals to be used in brain diagnosis using S.P.E.T. (Single Photon Emission Tomography). Initially, the rational planning had been performed with the new DADT molecular structures as radiopharmaceutical candidates. Using of Q.S.A.R. (Quantitative Structure Activity Relationship) techniques, the molecular descriptors such as partition coefficient and effective polarizability, have been studied in order to increase the blood brain barrier transport and the brain uptake respectively. Applying the Q.S.P.R. (Quantitative Structure Property Relationship) concepts to perform drug latentiation, based on bio-labile functional groups, the congener DADT derivative has been transformed into a pro-drug that works as a DADT moiety carrier, allowing the increasing of brain radiopharmaceutical uptake. Later on, synthetic routes and chemical purifications have been developed allowing the creation of the proposed chemical structure. Each new DADT derivative has been synthesized and analyzed in terms of elemental analysis, infrared and NMR spectra, in order to confirm its proposed chemical structure. Then, the new derivative has been labeled with 99mTc, radiochemically purified, intravenously injected in Swiss mice, allowing its biodistribution to evidence its brain transport and uptake. The rational planning studies have been re-evaluated after each biodistribution had been performed, to see what kind of molecular descriptor was responsible for causing a stronger optimization in the brain perfusion characteristics and then, new DADT derivatives have been prepared. Three new DADT derivatives have been obtained by using QSAR

  4. A Bacterial Virulence Protein Promotes Pathogenicity by Inhibiting the Bacterium's Own F1Fo ATP Synthase

    OpenAIRE

    Lee, Eun-Jin; Pontes, Mauricio H.; Groisman, Eduardo A.

    2013-01-01

    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 establis...

  5. Palmitate-induced changes in energy demand cause reallocation of ATP supply in rat and human skeletal muscle cells.

    Science.gov (United States)

    Nisr, Raid B; Affourtit, Charles

    2016-09-01

    Mitochondrial dysfunction has been associated with obesity-related muscle insulin resistance, but the causality of this association is controversial. The notion that mitochondrial oxidative capacity may be insufficient to deal appropriately with excessive nutrient loads is for example disputed. Effective mitochondrial capacity is indirectly, but largely determined by ATP-consuming processes because skeletal muscle energy metabolism is mostly controlled by ATP demand. Probing the bioenergetics of rat and human myoblasts in real time we show here that the saturated fatty acid palmitate lowers the rate and coupling efficiency of oxidative phosphorylation under conditions it causes insulin resistance. Stearate affects the bioenergetic parameters similarly, whereas oleate and linoleate tend to decrease the rate but not the efficiency of ATP synthesis. Importantly, we reveal that palmitate influences how oxidative ATP supply is used to fuel ATP-consuming processes. Direct measurement of newly made protein demonstrates that palmitate lowers the rate of de novo protein synthesis by more than 30%. The anticipated decrease of energy demand linked to protein synthesis is confirmed by attenuated cycloheximide-sensitivity of mitochondrial respiratory activity used to make ATP. This indirect measure of ATP turnover indicates that palmitate lowers ATP supply reserved for protein synthesis by at least 40%. This decrease is also provoked by stearate, oleate and linoleate, albeit to a lesser extent. Moreover, palmitate lowers ATP supply for sodium pump activity by 60-70% and, in human cells, decreases ATP supply for DNA/RNA synthesis by almost three-quarters. These novel fatty acid effects on energy expenditure inform the 'mitochondrial insufficiency' debate. PMID:27154056

  6. Endomorphin synthesis in rat brain from intracerebroventricularly injected [3H]-Tyr-Pro: a possible biosynthetic route for endomorphins.

    Science.gov (United States)

    Rónai, András Z; Szemenyei, Erzsébet; Kató, Erzsébet; Kocsis, László; Orosz, György; Al-Khrasani, Mahmoud; Tóth, Géza

    2006-03-15

    In spite of concentrated efforts, the biosynthetic route of mu-opioid receptor agonist brain tetrapeptide endomorphins (Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2), discovered in 1997, is still obscure. We report presently that 30 min after intracerebroventricular injection of 20 or 200 microCi [3H]Tyr-Pro (49.9 Ci mmol(-1)) the incorporated radioactivity was found in endomorphin-related tetra- and tripeptides in rat brain extracts. As detected by the combination of HPLC with radiodetection, a peak corresponding to endomorphin-2-OH could be identified in two of four extracts of "20 microCi" series. Radioactive peaks in position of Tyr, Tyr-Pro, Tyr-Pro-Phe or Tyr-Pro-Trp appeared regularly in both series and also in the "tetrapeptide cluster" constituted by endomorphins and their free carboxylic forms. In one of the four extracts in the "200 microCi" series a robust active peak in the position of endomorphin 2 could be detected. Intracerebroventricularly injected 100 nmol, but not 10 or 1000 nmol cold Tyr-Pro (devoid of opioid activity in vitro), caused a naloxone-reversible prolongation of tail-flick latency in rats, peaking between 15 and 30 min. We suggest that Tyr-Pro may serve as a biosynthetic precursor to endomorphin synthesis. PMID:16464510

  7. Intra-Blood-Brain Barrier Synthesis of Human Immunodeficiency Virus Antigen and Antibody in Humans and Chimpanzees

    Science.gov (United States)

    Goudsmit, Jaap; Epstein, Leon G.; Paul, Deborah A.; van der Helm, Hayo J.; Dawson, George J.; Asher, David M.; Yanagihara, Richard; Wolff, Axel V.; Gibbs, Clarence J.; Carleton Gajdusek, D.

    1987-06-01

    The presence of human immunodeficiency virus (HIV) antigens in cerebrospinal fluid (CSF) was associated with progressive encephalopathy in adult and pediatric patients with acquired immunodeficiency syndrome (AIDS). HIV antigen was detected in CSF from 6 of 7 AIDS patients with progressive encephalopathy. By contrast, HIV antigen, whether free or complexed, was detected in CSF from only 1 of 18 HIV antibody seropositive patients without progressive encephalopathy and from 0 of 8 experimentally infected chimpanzees without clinical signs. Intra-blood-brain barrier synthesis of HIV-specific antibody was demonstrated in the majority of patients with AIDS (9/12) or at risk for AIDS (8/13) as well as in the experimentally infected chimpanzees, indicating HIV-specific B-cell reactivity in the brain without apparent neurological signs. In 6 of 11 patients with HIV infection, antibodies synthesized in the central nervous system were directed against HIV envelope proteins. Active viral expression appears to be necessary for both the immunodeficiency and progressive encephalopathy associated with HIV infection.

  8. Synthesis and characterization of EADAM: a selective radioligand for mapping the brain serotonin transporters by positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Jarkas, Nachwa [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); McConathy, Jonathan [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Department of Psychiatry and Behavior Sciences, Emory University, Atlanta, GA 30322 (United States); Votaw, John R. [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Voll, Ronald J. [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Malveaux, Eugene [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Camp, Vernon M. [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Williams, Larry [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Goodman, Robin R. [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States); Kilts, Clinton D. [Department of Psychiatry and Behavior Sciences, Emory University, Atlanta, GA 30322 (United States); Goodman, Mark M. [Department of Radiology, Division of Radiological Sciences, Emory University, Atlanta, GA 30322 (United States) and Department of Psychiatry and Behavior Sciences, Emory University, Atlanta, GA 30322 (United States)]. E-mail: mgoodma@emory.edu

    2005-01-01

    [{sup 11}C]N,N-Dimethyl-2-(2'-amino-4'-ethylphenylthio)benzylamine ([{sup 11}C]EADAM) was synthesized in the development of a serotonin transporter (SERT) imaging ligand for positron emission tomography (PET). The methods of ligand synthesis, results of in vitro characterization, {sup 11}C labeling and in vivo micro-PET imaging studies of [{sup 11}C]EADAM in cynomolgus monkey brain are described. {sup 11}C was introduced into N,N-dimethyl-2-(2'-amino-4'-ethylphenylthio)benzylamine () by alkylation of N-methyl-2-(2'-amino-4'-ethylphenylthio)benzylamine () in 32% radiochemical yield (end of bombardment [EOB], decay-corrected from [{sup 11}C]methyl iodide). Competition binding assays in cells stably expressing the transfected human dopamine transporter (DAT), SERT and norepinephrine transporter (NET) labeled with [{sup 3}H]WIN 35428 or [{sup 125}I]RTI-55, [{sup 3}H]citalopram and [{sup 3}H]nisoxetine, respectively, indicated the following order of SERT affinity: ADAM>EADAM>>fluvoxamine. The affinity of EADAM for DAT and NET was 500- and >1000-fold lower, respectively, than for SERT. Micro-PET brain imaging studies in a cynomolgus monkey demonstrated high [{sup 11}C]EADAM uptake in the striatum, thalamus and brainstem. [{sup 11}C]EADAM uptake in these brain regions peaked in less than 60 min following administration of [{sup 11}C]EADAM. The tissue-to-cerebellum ratios of the striatum, thalamus and brainstem were 1.67, 1.71 and 1.63, respectively, at 120 min postinjection of [{sup 11}C]EADAM. Analysis of monkey arterial plasma samples using high-pressure liquid chromatography determined there was no detectable formation of lipophilic radiolabeled metabolites capable of entering the brain. In a displacement experiment with citalopram in a cynomolgus monkey, radioactivity in the striatum, thalamus and brainstem was displaced 20-60 min after administration of citalopram. In a blocking experiment with citalopram in a cynomolgus monkey

  9. Asymmetric Synthesis of Spirocyclic 2-Benzopyrans for Positron Emission Tomography of σ1 Receptors in the Brain

    Directory of Open Access Journals (Sweden)

    Katharina Holl

    2014-01-01

    Full Text Available Sharpless asymmetric dihydroxylation of styrene derivative 6 afforded chiral triols (R-7 and (S-7, which were cyclized with tosyl chloride in the presence of Bu2SnO to provide 2-benzopyrans (R-4 and (S-4 with high regioselectivity. The additional hydroxy moiety in the 4-position was exploited for the introduction of various substituents. Williamson ether synthesis and replacement of the Boc protective group with a benzyl moiety led to potent σ1 ligands with high σ1/σ2-selectivity. With exception of the ethoxy derivative 16, the (R-configured enantiomers represent eutomers with eudismic ratios of up to 29 for the ester (R-18. The methyl ether (R-15 represents the most potent σ1 ligand of this series of compounds, with a Ki value of 1.2 nM and an eudismic ratio of 7. Tosylate (R-21 was used as precursor for the radiosynthesis of [18F]-(R-20, which was available by nucleophilic substitution with K[18F]F K222 carbonate complex. The radiochemical yield of [18F]-(R-20 was 18%–20%, the radiochemical purity greater than 97% and the specific radioactivity 175–300 GBq/µmol. Although radiometabolites were detected in plasma, urine and liver samples, radiometabolites were not found in brain samples. After 30 min, the uptake of the radiotracer in the brain was 3.4% of injected dose per gram of tissue and could be reduced by coadministration of the σ1 antagonist haloperidol. [18F]-(R-20 was able to label those regions of the brain, which were reported to have high density of σ1 receptors.

  10. Premitotic DNA synthesis in the brain of the adult frog (Rana esculenta L.): An autoradiographic 3H-thymidine study

    International Nuclear Information System (INIS)

    Replicative synthesis of DNA in the brain of the adult frog was studied by light microscope autoradiography. Animals collected during the active period (May-June) and in hibernation (January) were used. In active frogs, 3H-thymidine labelling occurred mainly in the ependymal cells which line the ventricles. The mean labelling index (LI%) was higher in the ependyma of the lateral and fourth ventricles than in the ependyma of the lateral diencephalon and tectal parts of the mesencephalon. In the recessus infundibularis and preopticus the number of labelled cells (LCs) was several times greater than in the lateral parts of the third ventricle. LCs were seen subependymally only occasionally. The incidence of LCs in the parenchyma of the brain was much lower in most regions than in the ventricular ependyma; LCs were mainly small and, from their nuclear morphology, they were glial cells. The LI% reached the highest value in the septum hippocampi and in the nucleus entopeduncularis. In these locations, LCs were larger and closer in size to the nerve cells of these regions. From comparison with data obtained earlier in the brain of mammals, it is evident that the distribution of proliferating cells in the olfactory and limbic system is phylogenetically conservative. The occurrence of pyknotic cells in the same areas which contain LCs, suggests that cell division reflects in part the process of cell renewal observed in mammals. However, proliferating cells could also be linked to the continuous growth observed in non-mammalian vertebrates. In hibernating frogs, LCs and pyknoses were not seen or were found occasionally, which further indicates the functional significance of both processes

  11. Synthesis of [11C]citalopram and brain distribution studies in rats

    International Nuclear Information System (INIS)

    The study of serotonin uptake sites in the living human brain by PET with [11C]citalopram may be valuable in investigating the anatomic locus and the therapeutic role of depression and prevention of suicide. For this purpose, the authors have synthesized [11C]citalopram. In vivo biodistribution in rats has been determined

  12. A futile cycle, formed between two ATP-dependant -glutamyl cycle enzymes, -glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

    Indian Academy of Sciences (India)

    Akhilesh Kumar; Anand Kumar Bachhawat

    2010-03-01

    Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the -glutamyl cycle leads to ATP depletion. The enzyme -glutamyl cysteine synthetase (-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, -glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

  13. Clathrin-coated vesicles contain an ATP-dependent proton pump.

    OpenAIRE

    Forgac, M; Cantley, L; Wiedenmann, B; Altstiel, L; Branton, D

    1983-01-01

    Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+ and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a delta pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles. ATP-dependent [14C]methylamine uptake was abolished by the proton ionophore c...

  14. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  15. Effect of L-carnitine on postischemic inhibition of protein synthesis in the rat brain.

    Science.gov (United States)

    Burda, Jozef; Hernández Viadel, Mariluz; Danielisová, Viera; Némethová, Miroslava; Montoliu, Carmina; Felipo, Vicente

    2009-09-01

    The purpose of this study was to investigate effects of carnitine administration on protein synthesis recovery after transient cerebral ischemia. Rats received L-carnitine in two doses of 16 mmol/kg i.p. 15 min before ischemia and just on the onset of reperfusion. Transient forebrain ischemia was induced by 4-vessel occlusion for 15 min, followed by 30 min or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. A protective effect was observed, on protein synthesis as well as the number of surviving neurons, in the L-carnitine-treated groups. Our results indicate that L-carnitine can exert a protective effect in the development of reperfusion-induced injury. L-carnitine significantly reduced the ischemia/reperfusion-induced inhibition of translation and neurodegeneration in the neocortex as well as in the highly sensitive hippocampus and dorsolateral striatum. We expect that the ability of L-carnitine to keep translational machinery on facilitates efficacy of postischemic remodulation of gene expression. PMID:20037189

  16. Protein synthesis evaluation in brain and other organs in human by PET

    International Nuclear Information System (INIS)

    The choice of treatment in diseases of the nervous system cannot be based only on symptomatology, but on a presumed underlying pathological state. These pathological states often involve direct modifications of neuronal metabolism. Two areas of cellular biochemistry can be studied in vivo in humans: 1) glucose or oxygen consumption which is mainly responsible for energy and lipid metabolism. 2) amino acid metabolism, which is involved in protein and neurotransmitter synthesis. Here the authors examine protein synthesis, which is the basis of cellular integrity and tissue structure. Study of protein synthesis (PS) by positron emission tomography (PET) is governed by specific requirements dictated by 1) the metabolic pathways we want to explore (the fate of the tracer directly influences the analysis of the results); 2) The construction and validation of a mathematical model to be applied to the computerized images; and 3) the human pathology being studied. The timing of scanning and the experimental protocol must include in their conception some physiological constraints such as volume of organs, rapidity of biological phenomena, etc. All these steps are detailed in the following paragraphs

  17. Effect of brain-derived neurotrophic factor on BV2 microglia activated by ATP%脑源性神经营养因子在ATP活化BV2小胶质细胞中的作用

    Institute of Scientific and Technical Information of China (English)

    金海祥; 吴周浩

    2016-01-01

    目的:探讨脑源性神经营养因子(BDNF)在腺苷三磷酸(ATP)激活BV2小胶质细胞过程中的作用。方法以不同浓度ATP孵育BV2小胶质细胞后,采用Western blot法定量检测细胞中CD11b、BDNF表达水平,ELISA测定上清液中肿瘤坏死因子α(TNF‐α)的分泌水平。再将BV2细胞用不同浓度BDNF清除剂原肌球蛋白相关激酶B(TrkB)/Fc预处理后给予ATP孵育,检测细胞中CD11b、BDNF表达水平的变化及上清液中 TNF‐α的分泌水平。最后加入外源性重组BDNF ,检测细胞内CD11b表达和上清液中TNF‐α的水平变化。结果 ATP孵育BV2细胞后,细胞内 CD11b、BDNF及上清液中 TNF‐α水平在一定范围内呈剂量时间依赖性增加。加入TrkB/Fc后,BV2细胞中 CD11b、BDNF及上清液中TNF‐α表达水平在一定范围内呈剂量和时间依赖性降低。加入外源性BDNF后,细胞内CD11b及上清液中TNF‐α水平又出现增加。结论 BV2小胶质细胞活化后细胞内BDNF增多,外源性补充BDNF可激活小胶质细胞,BDNF在小胶质细胞的活化过程中可能发挥了重要作用。%Objective To study the effect of brain‐derived neurotrophic factor (BDNF) in the process of adenosine triphos‐phate (ATP) activating BV2 microglia .Methods BV2 microglia was cultured by adding different concentrations of ATP .Then the expression level of intracellular CD11b and BDNF and the secretion level of TNF‐α in the supernatant were quantitatively deter‐mined by Western blot .BV2 microglia was treated by different concentrations of BDNF scavenger tyrosine kinase receptors B (TrkB)/Fc and incubated by ATP .The expression level of intracellular CD11b and BDNF and the secretion level of TNF‐αin the supernatant were measured .Finally adding exogenous recombinant BDNF into cultured BV 2 microglia ,intracellular changes of CD11b and supernatant TNF‐αlevels were detected .Results After adding ATP for cultivating BV2

  18. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Amstrup, Jan; Henriksen, Katrine Lütken;

    2003-01-01

    ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock...

  19. Rapid synthesis and purification of carbon-11 labelled DOPA: a potential agent for brain studies

    International Nuclear Information System (INIS)

    A rapid method for preparation and purification of β-(3,4-dihydroxyphenyl)-D,L-α-alanine-1-11C(11C-DOPA), using 11CO2 as the radioactive precursor is described. Carboxylation of an α-lithioisocyanide, containing protected hydroxylic groups, was followed by a three-step hydrolysis of the intermediate αioscyano carboxylic acid. Preliminary experiments in rats indicate that the compound is preferentially decarboxylated in brain areas rich in dopamine containing neurons. (author)

  20. Dynamics of endogenous Hsp70 synthesis in the brain of olfactory bulbectomized mice

    OpenAIRE

    Bobkova, Natalia; Guzhova, Irina; Margulis, Boris; Nesterova, Inna; Medvedinskaya, Natalia; Samokhin, Alexander; Alexandrova, Irina; Garbuz, David; Nudler, Evgeny; Evgen’ev, Michael

    2012-01-01

    Numerous epidemiological studies have established acute brain injury as one of the major risk factors for the Alzheimer's disease (AD). However, the lack of animal models of AD-like degeneration triggered by a defined injury hampered the development of adequate therapies. Here we report that the surgical damage of the olfactory bulbs triggers the development of several pathologies, including amyloid-β accumulation and strong decrease of neuron density in the cortex and hippocampus as well as ...

  1. Metabolism of choline in brain of the aged CBF-1 mouse

    International Nuclear Information System (INIS)

    In order to quantify the changes that occur in the cholinergic central nervous system with aging, we have compared acetylcholine (Ach) formation in brain cortex slice preparations from 2-year-old aged CBF-1 mouse brains and compared the findings with those in 2-4-month-old young adult mouse brain slices. Incorporation of exogenous radioactively labelled choline (31 nM [3H] choline) into acetyl choline in incubated brain slices was linear with time for 90 min. Percentage of total choline label distributed into Ach remained constant from 5 min after starting the incubation to 90 min. In contrast, distribution of label into intracellular free choline (Ch) and phosphorylcholine (Pch) changed continuously over this period suggesting that the Ch pool for Ach synthesis in brain cortex is different from that for Pch synthesis. Incorporation of radioactivity into Ach was not influenced by administration of 10 microM eserine, showing that the increment of radioactivity in Ach reflects rate of Ach formation, independently from degradation by acetylcholine esterases. Under our experimental conditions, slices from cortices of aged 24-month-old mouse brain showed a significantly greater (27%) incorporation of radioactivity into intracellular Ach than those from young, 2-4-month-old, brain cortices. Inhibitors of Ach release, 1 mM ATP or GABA, had no effect. Since concentration of radioactive precursor in the incubation medium was very low (31 nM), the Ch pool for Ach synthesis in slices was labelled without measurably changing the size of the endogenous pool. These data suggest a compensatory acceleration of Ach synthesis or else a smaller precursor pool specific for Ach synthesis into which labelled Ch migrated in aged brain

  2. Decline of Phosphotransfer and Substrate Supply Metabolic Circuits Hinders ATP Cycling in Aging Myocardium.

    Directory of Open Access Journals (Sweden)

    Emirhan Nemutlu

    Full Text Available Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK, creatine kinase (CK, and glycolytic/glycogenolytic pathways using 18O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to 18O-labeling procedure at resting basal state, and analyzed using the 18O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[18O], γ-ATP[18O], β-ADP[18O], and creatine phosphate CrP[18O] 18O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[18O] turnover with relatively constant glycogenolytic flux or G1P[18O] 18O-labeling. Labeling of G3P[18O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of 18O into second (18O2, third (18O3, and fourth (18O4 positions of Pi[18O] and a lower Pi[18O]/γ-ATP[18 O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, 18O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic

  3. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    OpenAIRE

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  4. Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

    OpenAIRE

    Hayoz Sébastien; Jia Cuihong; Hegg CC

    2012-01-01

    Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results...

  5. Improved mitochondrial function in brain aging and Alzheimer disease - the new mechanism of action of the old metabolic enhancer piracetam

    Directory of Open Access Journals (Sweden)

    Kristina Leuner

    2010-09-01

    Full Text Available Piracetam, the prototype of the so-called nootropic drugs’ is used since many years in different countries to treat cognitive impairment in aging and dementia. Findings that piracetam enhances fluidity of brain mitochondrial membranes led to the hypothesis that piracetam might improve mitochondrial function, e.g. might enhance ATP synthesis. This assumption has recently been supported by a number of observations showing enhanced mitochondrial membrane potential (MMP, enhanced ATP production, and reduced sensitivity for apoptosis in a variety of cell and animal models for aging and Alzheimer disease (AD. As a specific consequence, substantial evidence for elevated neuronal plasticity as a specific effect of piracetam has emerged. Taken together, these new findings can explain many of the therapeutic effects of piracetam on cognition in aging and dementia as well as different situations of brain dysfunctions.

  6. Synthesis of iodine-123 labeled 3-O-(E)-3-iodopropen-2-yl-D-glucose: A potential new agent for the assessment of glucose transport into the brain and heart using SPECT

    International Nuclear Information System (INIS)

    The authors have developed a synthesis of iodine-123 labelled 3-O-(E)-3-iodopropen-2-yl-D-glucose as an analogue of carbon-11 labelled 3-O-methyl-D-glucose, with the objective of achieving high brain and heart uptake. The four reaction steps in the synthesis are discussed and tissue distributions in rats will be presented

  7. ATP independent and ATP dependent chromatin remodeling in wheat

    International Nuclear Information System (INIS)

    Unraveling the biochemistry of chromatin dynamics during DNA replication, repair, recombination as well as transcription is the current challenge in biology. The nucleosomes containing histone octamer are the crucial elements responsible for winding and unwinding eukaryotic DNA. During DNA centric events, these nucleosomes translocate along the DNA with concomitant covalent modifications of histones. We explored these mechanisms in wheat seedlings after irradiation with survivable dose of 60Co-γ radiations. The histones isolated from irradiated seedlings showed that global acetylation of H3 decreased and H4 increased in dose depend manner till 100 grays. Time course of individual modifications showed that for H3K4 and H3K9 acetylation decreased, whereas H3S10, phosphorylation increased. There were fluctuations in acetylation of H4K5, H4K12 and H4K16, whereas H4K8 showed hyperacetylation. We found ATP-dependent chromatin remodeling activity as trans-transfer of the nucleosomes from wheat native donor chromatin on a labeled nucleosome positioning sequence and cis-transfer of the mononucleosomes in vitro. However, there was no significant change in this activity in extracts obtained from irradiated wheat seedlings. This is the first report on, demonstration of ATP-dependent chromatin remodeling activity and site specific H3 and H4 modifications in response to exposure to ionizing radiation in case of plants. (author)

  8. Parallel synthesis of a series of potentially brain penetrant aminoalkyl benzoimidazoles.

    Science.gov (United States)

    Micco, Iolanda; Nencini, Arianna; Quinn, Joanna; Bothmann, Hendrick; Ghiron, Chiara; Padova, Alessandro; Papini, Silvia

    2008-03-01

    Alpha7 agonists were identified via GOLD (CCDC) docking in the putative agonist binding site of an alpha7 homology model and a series of aminoalkyl benzoimidazoles was synthesised to obtain potentially brain penetrant drugs. The array was prepared starting from the reaction of ortho-fluoronitrobenzenes with a selection of diamines, followed by reduction of the nitro group to obtain a series of monoalkylated phenylene diamines. N,N'-Carbonyldiimidazole (CDI) mediated acylation, followed by a parallel automated work-up procedure, afforded the monoacylated phenylenediamines which were cyclised under acidic conditions. Parallel work-up and purification afforded the array products in good yields and purities with a robust parallel methodology which will be useful for other libraries. Screening for alpha7 activity revealed compounds with agonist activity for the receptor. PMID:18078760

  9. Asymmetry of rotational catalysis of single membrane-bound F0F1-ATP synthase

    CERN Document Server

    Zarrabi, Nawid; Diez, Manuel; Graeber, Peter; Wrachtrup, Joerg; Boersch, Michael

    2007-01-01

    Synthesis of the cellular 'energy currency' ATP is catalyzed by membrane-bound F0F1-ATP synthases. The chemical reaction at three binding sites in the F1 part is coupled to proton translocation through the membrane-integrated F0 part by an internal rotation of subunits. We examined the rotary movements of the epsilon-subunit of the 'rotor' with respect to the b-subunits of the 'stator' by single-molecule fluorescence resonance energy transfer (FRET). Rotation of epsilon during ATP hydrolysis is divided into three major steps with constant FRET level corresponding to three binding sites. Different catalytic activities of the individual binding sites were observed depending on the relative orientation of the 'rotor'. Computer simulations of the FRET signals and non-equally distributed orientations of epsilon strongly corroborate asymmetry of catalysis in F0F1-ATP synthase.

  10. Peripheral SLC6A4 DNA methylation is associated with in vivo measures of human brain serotonin synthesis and childhood physical aggression.

    Directory of Open Access Journals (Sweden)

    Dongsha Wang

    Full Text Available The main challenge in addressing the role of DNA methylation in human behaviour is the fact that the brain is inaccessible to epigenetic analysis in living humans. Using positron emission tomography (PET measures of brain serotonin (5-HT synthesis, we found in a longitudinal sample that adult males with high childhood-limited aggression (C-LHPA had lower in vivo 5-HT synthesis in the orbitofrontal cortex (OBFC. Here we hypothesized that 5-HT alterations associated with childhood aggression were linked to differential DNA methylation of critical genes in the 5-HT pathway and these changes were also detectable in peripheral white blood cells. Using pyrosequencing, we determined the state of DNA methylation of SLC6A4 promoter in T cells and monocytes isolated from blood of cohort members (N = 25 who underwent a PET scan, and we examined whether methylation status in the blood is associated with in vivo brain 5-HT synthesis. Higher levels of methylation were observed in both T cells and monocytes at specific CpG sites in the C-LHPA group. DNA methylation of SLC6A4 in monocytes appears to be associated more reliably with group membership than T cells. In both cell types the methylation state of these CpGs was associated with lower in vivo measures of brain 5-HT synthesis in the left and right lateral OBFC (N = 20 where lower 5-HT synthesis in C-LHPA group was observed. Furthermore, in vitro methylation of the SLC6A4 promoter in a luciferase reporter construct suppresses its transcriptional activity supporting a functional role of DNA methylation in SLC6A4 promoter regulation. These findings indicate that state of SLC6A4 promoter methylation is altered in peripheral white blood cells of individuals with physical aggression during childhood. This supports the relevance of peripheral DNA methylation for brain function and suggests that peripheral SLC6A4 DNA methylation could be a marker of central 5-HT function.

  11. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution......, since the method is very sensitive (detects 0.5 ng ATP/L) and results are obtained within minutes. When calculating the ATP value a number of parameters need to be considered. These were investigate by use of two different reagent kits (PCP-kit and Lumin(ATE)/Lumin(EX)-kit), internal standard and an...... Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...

  12. Protein targeting to ATP-dependent proteases

    OpenAIRE

    Inobe, Tomonao; Matouschek, Andreas

    2008-01-01

    ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be u...

  13. Autoradiographic studies of the rate of DNA synthesis in the rat epididymis duct epithelium and brain subependimal zone cells after the whole body X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Gracheva, N.D.; Shatalin, G.I. (Tsentral' nyj Nauchno-Issledovatel' skij Rentgeno-Radiologicheskij Inst., Leningrad (USSR))

    1982-11-01

    DNA synthesis rate was analyzed on the basis of labelled cell distribution in epithelium of epididymis duct and subependyma zone of rat brain from the number of reduced silver grains under a nucleus calculated on recorders of histologic sections (5 ..mu..m) during different time after /sup 3/H hymidine intake and total X-ray irradiation in 300 Gy dose. Results of observations served as the additional substation of an earlier conclusion that in a series of truncal-semitruncal-differentiated cell per stage decrease of DNA synthesis rate occurs. During the period of maximum postradiation repair the proliferation increase took place at the expense of cell self-reproducibility, which in norm have medium and high rates of DNA synthesis against the background of cell preproduction deceleration which are characterized in norm with low rates of DNA synthesis and after mitosis should initiate differentiation. These facts conditioned the increase in the mean number of the reduced silver grains per a nucleus at a height of the postradiation proliferation, while DNA synthesis rates themselves peculiar to successive generations of truncal cells didn't change.

  14. Synthesis of ( sup 11 C)citalopram and brain distribution studies in rats

    Energy Technology Data Exchange (ETDEWEB)

    Ram, S.; Krishnan, K.R.R.; Bissette, G.; Knight, D.L.; Coleman, R.E. (Duke Univ., Durham, NC (USA). Medical Center)

    1991-01-01

    Citalopram (1-(3-dimethylamino)propyl-1-(p-fluoro-phenyl)-5-phthalancarbonitrile) is a selective serotonin uptake inhibitor, and this prototype drug possesses high affinity for serotonin uptake sites and is used in the treatment of depression. We have synthesized ({sup 11}C)citalopram by alkylation. The procedure involves the reaction of ({sup 11}C)iodomethane with desmethylcitalopram in acetone in the presence of sodium hydroxide base at 65{sup o}C for 8-10 min, and followed by purification by column, which contained, in series silica gel and basic alumina produces pure ({sup 11}C)citalopram with a specific activity of 150-434 Ci/mmol (at EOS). The radiochemical yields were 18% to 66% (at EOB), with a radiochemical purity range 92% to 99%. In vivo biodistribution of ({sup 11}C)citalopram in Sprague-Dawley rats brain clearly differentiates regions of high (frontal cortex, substantia niagra and hypothalamus) and low (cerebellum) uptake corresponding to the known distribution of serotonin uptake in rats and primates. These results demonstrate that this ligand is suitable for study of serotonin uptake sites by P.E.T., and may be useful as a biochemical diagnostic imaging tool in various psychiatric disorders. Further studies with this ligand are in progress. (author).

  15. Modulation of Extracellular ATP Content of Mast Cells and DRG Neurons by Irradiation: Studies on Underlying Mechanism of Low-Level-Laser Therapy

    Directory of Open Access Journals (Sweden)

    Lina Wang

    2015-01-01

    Full Text Available Low-level-laser therapy (LLLT is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca2+]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

  16. Cooperation and competition between adenylate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 31P-nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport

  17. Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation

    Science.gov (United States)

    Bojovschi, A.; Liu, Ming S.; Sadus, Richard J.

    2012-08-01

    The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F1-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg2+ leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

  18. A wireless brain-machine interface for real-time speech synthesis.

    Directory of Open Access Journals (Sweden)

    Frank H Guenther

    Full Text Available BACKGROUND: Brain-machine interfaces (BMIs involving electrodes implanted into the human cerebral cortex have recently been developed in an attempt to restore function to profoundly paralyzed individuals. Current BMIs for restoring communication can provide important capabilities via a typing process, but unfortunately they are only capable of slow communication rates. In the current study we use a novel approach to speech restoration in which we decode continuous auditory parameters for a real-time speech synthesizer from neuronal activity in motor cortex during attempted speech. METHODOLOGY/PRINCIPAL FINDINGS: Neural signals recorded by a Neurotrophic Electrode implanted in a speech-related region of the left precentral gyrus of a human volunteer suffering from locked-in syndrome, characterized by near-total paralysis with spared cognition, were transmitted wirelessly across the scalp and used to drive a speech synthesizer. A Kalman filter-based decoder translated the neural signals generated during attempted speech into continuous parameters for controlling a synthesizer that provided immediate (within 50 ms auditory feedback of the decoded sound. Accuracy of the volunteer's vowel productions with the synthesizer improved quickly with practice, with a 25% improvement in average hit rate (from 45% to 70% and 46% decrease in average endpoint error from the first to the last block of a three-vowel task. CONCLUSIONS/SIGNIFICANCE: Our results support the feasibility of neural prostheses that may have the potential to provide near-conversational synthetic speech output for individuals with severely impaired speech motor control. They also provide an initial glimpse into the functional properties of neurons in speech motor cortical areas.

  19. Continuous intravenous infusion of ATP in humans yields large expansions of erythrocyte ATP pools but extracellular ATP pools are elevated only at the start followed by rapid declines

    OpenAIRE

    Rapaport, Eliezer; Salikhova, Anna; Abraham, Edward H.

    2015-01-01

    The pharmacokinetics of adenosine 5′-triphosphate (ATP) was investigated in a clinical trial that included 15 patients with advanced malignancies (solid tumors). ATP was administered by continuous intravenous infusions of 8 h once weekly for 8 weeks. Three values of blood ATP levels were determined. These were total blood (erythrocyte) and blood plasma (extracellular) ATP pools along with the initial rate of release of ATP into the blood plasma. We found that values related to erythrocyte ATP...

  20. The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    Science.gov (United States)

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion. PMID:27482602

  1. Fluorogenic ATP Analogues for Online Monitoring of ATP Consumption : Observing Ubiquitin Activation in Real Time

    OpenAIRE

    Hacker, Stephan; Pagliarini, Dana; Tischer, Thomas; Hardt, Normann; Schneider, Daniel; Mex, Martin; Mayer, Thomas; Scheffner, Martin; Marx, Andreas

    2013-01-01

    Many enzymes use ATP in signal-transducing processes or as an energy source. New fluorogenic ATP analogues signal ATP consumption by ubiquitin-like protein-activating enzymes in real time. Thus the inhibition and stimulation of these ATP-processing enzymes can be studied without auxiliary enzymes and reagents. beta-Lapachone was identified as an inhibitor of the ubiquitin-activating enzyme UBA1 (see scheme; A=acceptor, D=donor).

  2. Facile large-scale synthesis of brain-like mesoporous silica nanocomposites via a selective etching process

    Science.gov (United States)

    Chen, Yu; Wang, Qihua; Wang, Tingmei

    2015-10-01

    The core-shell structured mesoporous silica nanomaterials (MSNs) are experiencing rapid development in many applications such as heterogeneous catalysis, bio-imaging and drug delivery wherein a large pore volume is desirable. We develop a one-pot method for large-scale synthesis of brain-like mesoporous silica nanocomposites based on the reasonable change of the intrinsic nature of the -Si-O-Si- framework of silica nanoparticles together with a selective etching strategy. The as-synthesized products show good monodispersion and a large pore volume of 1.0 cm3 g-1. The novelty of this approach lies in the use of an inorganic-organic hybrid layer to assist the creation of large-pore morphology on the outermost shell thereby promoting efficient mass transfer or storage. Importantly, the method is reliable and grams of products can be easily prepared. The morphology on the outermost silica shell can be controlled by simply adjusting the VTES-to-TEOS molar ratio (VTES: triethoxyvinylsilane, TEOS: tetraethyl orthosilicate) as well as the etching time. The as-synthesized products exhibit fluorescence performance by incorporating rhodamine B isothiocyanate (RITC) covalently into the inner silica walls, which provide potential application in bioimaging. We also demonstrate the applications of as-synthesized large-pore structured nanocomposites in drug delivery systems and stimuli-responsive nanoreactors for heterogeneous catalysis.The core-shell structured mesoporous silica nanomaterials (MSNs) are experiencing rapid development in many applications such as heterogeneous catalysis, bio-imaging and drug delivery wherein a large pore volume is desirable. We develop a one-pot method for large-scale synthesis of brain-like mesoporous silica nanocomposites based on the reasonable change of the intrinsic nature of the -Si-O-Si- framework of silica nanoparticles together with a selective etching strategy. The as-synthesized products show good monodispersion and a large pore volume

  3. Formation of peptides from amino acids by single or multiple additions of ATP to suspensions of nucleoproteinoid microparticles

    Science.gov (United States)

    Nakashima, T.; Fox, S. W.

    1981-01-01

    The synthesis of peptides from individual amino acids or pairs of amino acids and ATP in the presence of catalysis by nucleoproteinoid microparticles is investigated. Experiments were performed with suspensions formed from the condensation of lysine-rich and acidic proteinoids with polyadenylic acid, to which were added glycine, phenylalanine, proline, lysine or glycine-phenylalanine mixtures, and ATP either at once or serially. Peptide yields are found to be greatest for equal amounts of acidic and basic proteinoids. The addition of imidazole is found to alter the preference of glycine-phenylalanine mixtures to form mixed heteropeptides rather than homopeptides. A rapid ATP decay in the peptide synthesis reaction is observed, and a greater yield is obtained for repeated small additions than for a single addition of ATP. The experimental system has properties similar to modern cells, and represents an organizational unit ready for the evolution of associated biochemical pathways.

  4. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

    Full Text Available Abstract There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx or out (efflux of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]. ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders.

  5. Study of the Five Rickettsia prowazekii Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides

    OpenAIRE

    Audia, Jonathon P.; Winkler, Herbert H.

    2006-01-01

    The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interes...

  6. Huperzine A protects isolated rat brain mitochondria against beta-amyloid peptide.

    Science.gov (United States)

    Gao, Xin; Zheng, Chun Yan; Yang, Ling; Tang, Xi Can; Zhang, Hai Yan

    2009-06-01

    Our previous work in cells and animals showed that mitochondria are involved in the neuroprotective effect of huperzine A (HupA). In this study, the effects of HupA on isolated rat brain mitochondria were investigated. In addition to inhibiting the Abeta(25-35) (40 microM)-induced decrease in mitochondrial respiration, adenosine 5'-triphosphate (ATP) synthesis, enzyme activity, and transmembrane potential, HupA (0.01 or 0.1 microM) effectively prevented Abeta-induced mitochondrial swelling, reactive oxygen species increase, and cytochrome c release. More interestingly, administration of HupA to isolated mitochondria promoted the rate of ATP production and blocked mitochondrial swelling caused by normal osmosis. These results indicate that HupA protects mitochondria against Abeta at least in part by preserving membrane integrity and improving energy metabolism. These direct effects on mitochondria further extend the noncholinergic functions of HupA. PMID:19272446

  7. Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells

    International Nuclear Information System (INIS)

    Chinese hamster ovary (CHO-K1) cells were pulse labeled with [3H]serine, and the synthesis of phosphatidyl[3H]ethanolamine from phosphatidyl[3H]serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 μg of saponin per ml, there was no significant turnover of nascent phosphatidyl[3H]serine to form phosphatidyl[3H]ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl[3H]ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl[3H]serine during a subsequent 2-hr chase. Phosphatidyl[3H]ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl[3H]ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl[3H]serine to phosphatidyl[3H]ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5'-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl[3H]ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins

  8. Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells

    Energy Technology Data Exchange (ETDEWEB)

    Voelker, D.R. (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO (USA))

    1989-12-01

    Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({sup 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.

  9. ATP-triggered anticancer drug delivery

    Science.gov (United States)

    Mo, Ran; Jiang, Tianyue; Disanto, Rocco; Tai, Wanyi; Gu, Zhen

    2014-03-01

    Stimuli-triggered drug delivery systems have been increasingly used to promote physiological specificity and on-demand therapeutic efficacy of anticancer drugs. Here we utilize adenosine-5'-triphosphate (ATP) as a trigger for the controlled release of anticancer drugs. We demonstrate that polymeric nanocarriers functionalized with an ATP-binding aptamer-incorporated DNA motif can selectively release the intercalating doxorubicin via a conformational switch when in an ATP-rich environment. The half-maximal inhibitory concentration of ATP-responsive nanovehicles is 0.24 μM in MDA-MB-231 cells, a 3.6-fold increase in the cytotoxicity compared with that of non-ATP-responsive nanovehicles. Equipped with an outer shell crosslinked by hyaluronic acid, a specific tumour-targeting ligand, the ATP-responsive nanocarriers present an improvement in the chemotherapeutic inhibition of tumour growth using xenograft MDA-MB-231 tumour-bearing mice. This ATP-triggered drug release system provides a more sophisticated drug delivery system, which can differentiate ATP levels to facilitate the selective release of drugs.

  10. ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain

    International Nuclear Information System (INIS)

    The histidine WalK (YycG) plays a crucial role in coordinating murein synthesis with cell division and the crystal structure of its ATP binding domain has been determined. Interestingly the bound ATP was not hydrolyzed during crystallization and remains intact in the crystal lattice. In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferring the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg2+ ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3′ of the sugar ring and O2B of the β-phosphate, implying an internal hydrogen bond. The stability of the WalK–ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth

  11. ATP and Presentation Service for Mizar Formalizations

    CERN Document Server

    Urban, Josef; Sitcliffe, Geoff

    2011-01-01

    This paper describes the Automated Reasoning for Mizar (MizAR) service, which integrates several automated reasoning, artificial intelligence, and presentation tools with Mizar and its authoring environment. The service provides ATP assistance to Mizar authors in finding and explaining proofs, and offers generation of Mizar problems as challenges to ATP systems. The service is based on a sound translation from the Mizar language to that of first-order ATP systems, and relies on the recent progress in application of ATP systems in large theories containing tens of thousands of available facts. We present the main features of MizAR services, followed by an account of initial experiments in finding proofs with the ATP assistance. Our initial experience indicates that the tool offers substantial help in exploring the Mizar library and in preparing new Mizar articles.

  12. F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft

    Science.gov (United States)

    Kulish, O.; Wright, A. D.; Terentjev, E. M.

    2016-06-01

    F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the ‘no-load’ angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of ‘stall torque’. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the ‘useful outcome’ is measured in the number of H+ pushed against the chemical gradient.

  13. Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

    Science.gov (United States)

    Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

    2016-04-01

    Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit. PMID:26920481

  14. F1 rotary motor of ATP synthase is driven by the torsionally-asymmetric drive shaft

    Science.gov (United States)

    Kulish, O.; Wright, A. D.; Terentjev, E. M.

    2016-01-01

    F1F0 ATP synthase (ATPase) either facilitates the synthesis of ATP in a process driven by the proton moving force (pmf), or uses the energy from ATP hydrolysis to pump protons against the concentration gradient across the membrane. ATPase is composed of two rotary motors, F0 and F1, which compete for control of their shared γ -shaft. We present a self-consistent physical model of F1 motor as a simplified two-state Brownian ratchet using the asymmetry of torsional elastic energy of the coiled-coil γ -shaft. This stochastic model unifies the physical concepts of linear and rotary motors, and explains the stepped unidirectional rotary motion. Substituting the model parameters, all independently known from recent experiments, our model quantitatively reproduces the ATPase operation, e.g. the ‘no-load’ angular velocity is ca. 400 rad/s anticlockwise at 4 mM ATP. Increasing the pmf torque exerted by F0 can slow, stop and overcome the torque generated by F1, switching from ATP hydrolysis to synthesis at a very low value of ‘stall torque’. We discuss the motor efficiency, which is very low if calculated from the useful mechanical work it produces - but is quite high when the ‘useful outcome’ is measured in the number of H+ pushed against the chemical gradient. PMID:27321713

  15. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU)

    OpenAIRE

    Ronglan Zhao; Dongchun Liang; Deming Sun

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect...

  16. Knockdown of DAPIT (Diabetes-associated Protein in Insulin-sensitive Tissue) Results in Loss of ATP Synthase in Mitochondria

    OpenAIRE

    Ohsakaya, Shigenori; Fujikawa, Makoto; Hisabori, Toru; Yoshida, Masasuke

    2011-01-01

    It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of α-...

  17. Crystallization of the c[subscript 14]-rotor of the chloroplast ATP synthase reveals that it contains pigments

    Energy Technology Data Exchange (ETDEWEB)

    Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra (AZU)

    2008-08-27

    The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphage, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10--15 c-subunits is commonly thought to drive rotation of the rotor moiety (c{sub 10-14}{gamma}{sup {epsilon}}) relative to stator moiety ({alpha}{sub 3}{beta}{sub 3}{delta}ab{sub 2}). Here we report the isolation and crystallization of the c{sub 14}-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 {angstrom}. Though ATP synthase was not previously know to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revaled that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase.

  18. Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase.

    Science.gov (United States)

    Böckmann, Rainer A; Grubmüller, Helmut

    2002-03-01

    The mitochondrial membrane protein FoF1-ATP synthase synthesizes adenosine triphosphate (ATP), the universal currency of energy in the cell. This process involves mechanochemical energy transfer from a rotating asymmetric gamma-'stalk' to the three active sites of the F1 unit, which drives the bound ATP out of the binding pocket. Here, the primary structural changes associated with this energy transfer in F1-ATP synthase were studied with multi-nanosecond molecular dynamics simulations. By forced rotation of the gamma-stalk that mimics the effect of proton motive Fo-rotation during ATP synthesis, a time-resolved atomic model for the structural changes in the F1 part in terms of propagating conformational motions is obtained. For these, different time scales are found, which allows the separation of nanosecond from microsecond conformational motions. In the simulations, rotation of the gamma-stalk lowers the ATP affinity of the betaTP binding pocket and triggers fast, spontaneous closure of the empty betaE subunit. The simulations explain several mutation studies and the reduced hydrolysis rate of gamma-depleted F1-ATPase. PMID:11836535

  19. Matching ATP Supply and Demand in Mammalian Heart: In Vivo, In Vitro and In Silico Perspectives

    Science.gov (United States)

    Yaniv, Yael; Juhaszova, Magdalena; Nuss, H. Bradley; Wang, Su; Zorov, Dmitry B.; Lakatta, Edward G.; Sollott, Steven J.

    2010-01-01

    Although the heart rapidly adapts cardiac output to match the body’s circulatory demands, the regulatory mechanisms ensuring that sufficient ATP is available to perform the required cardiac work are not completely understood. Two mechanisms have been suggested to serve as key regulators: (1) ADP and Pi concentrations—ATP utilization/hydrolysis in the cytosol increases ADP and Pi fluxes to mitochondria and hence the amount of available substrates for ATP production increases; and (2) Ca2+ concentration—ATP utilization/hydrolysis is coupled to changes in free cytosolic calcium and mitochondrial calcium, the latter controlling Ca2+-dependent activation of mitochondrial enzymes taking part in ATP production. Here we discuss the evolving perspectives of each of the putative regulatory mechanisms and the precisemolecular targets (dehydrogenase enzymes, ATP synthase) based on existing experimental and theoretical evidence. The data synthesis can generate novel hypotheses and experimental designs to solve the ongoing enigma of energy supply–demand matching in the heart. PMID:20201896

  20. Design, synthesis, radiolabeling and in vivo evaluation of potential positron emission tomography (PET) radioligands for brain imaging of the 5-HT7 receptor

    DEFF Research Database (Denmark)

    Lacivita, Enza; Niso, Mauro; Hansen, Hanne D.; Di Pilato, Pantaleo; Herth, Matthias Manfred; Lehel, Szabolcs; Ettrup, Anders; Montenegro, Lisa; Perrone, Roberto; Berardi, Francesco; Colabufo, Nicola A.; Leopoldo, Marcello; Knudsen, Gitte M.

    2014-01-01

    Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds structurally related to the high affinity serotonin 5-HT7 receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (6, LP-211). Specific structural modifications were performed in or....... Positron emission tomography (PET) analysis revealed that [(11)C]-23a and [(11)C]-26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain uptake. These features were not predicted by in vitro tests....

  1. Brain regional {alpha}-[{sup 11}C]methyl-L-tryptophan trapping, used as an index of 5-HT synthesis, in healthy adults: absence of an age effect

    Energy Technology Data Exchange (ETDEWEB)

    Rosa-Neto, Pedro; Benkelfat, Chawki; Leyton, Marco [Department of Neurology and Neurosurgery, Montreal (Canada); McGill University, Department of Psychiatry, Montreal, Quebec (Canada); Sakai, Yojiro [Department of Neurology and Neurosurgery, Montreal (Canada); University of Tokyo, Department of Psychosomatic Medicine, Tokyo (Japan); Morais, Jose A. [McGill University, Department of Geriatrics, Montreal, Quebec (Canada); Diksic, Mirko [Department of Neurology and Neurosurgery, Montreal (Canada)

    2007-08-15

    Previous functional neuroimaging studies suggest that selective aspects of the brain serotonin (5-HT) system change during the aging process. Here, we assessed the effects of aging on the brain regional {alpha}-[{sup 11}C]methyl-L-tryptophan ({alpha}-[{sup 11}C]MTrp) trapping rate constant (K*; {mu}l.g{sup -1}.min{sup -1}), which, with certain assumptions, could be taken as a proxy of 5-HT synthesis. Thirty-six healthy right-handed subjects had positron emission tomography (PET) scans following injection with {alpha}-[{sup 11}C]MTrp [18 males aged 46.6 {+-} 22.2 years (range 20-80 years) and 18 females aged 33.0 {+-} 15.5 years (range 20-80 years)]. The trapping rate constant, K*, was calculated with the graphical method for irreversible ligands using the sinus-venous input function. A priori selected volumes of interest (VOIs) were defined using an automatic algorithm. VOI analysis showed no correlation between age and brain regional K* values. As reported by others, significant age-related reductions of gray matter were observed in the thalamus and frontal and cingulate cortices; even with partial volume correction there was still no significant relationship between K* and age. Further exploratory SPM voxelwise correlation between age and {alpha}-[{sup 11}C]MTrp trapping, using p = 0.05 (uncorrected), as well as voxel-based morphometry, was in agreement with the VOI analysis. The dissociation between age-related changes in brain anatomy and this index of serotonin synthesis suggests independent mechanisms underlying the normal aging process. (orig.)

  2. An ATP and oxalate generating variant tricarboxylic acid cycle counters aluminum toxicity in Pseudomonas fluorescens.

    Directory of Open Access Journals (Sweden)

    Ranji Singh

    Full Text Available Although the tricarboxylic acid (TCA cycle is essential in almost all aerobic organisms, its precise modulation and integration in global cellular metabolism is not fully understood. Here, we report on an alternative TCA cycle uniquely aimed at generating ATP and oxalate, two metabolites critical for the survival of Pseudomonas fluorescens. The upregulation of isocitrate lyase (ICL and acylating glyoxylate dehydrogenase (AGODH led to the enhanced synthesis of oxalate, a dicarboxylic acid involved in the immobilization of aluminum (Al. The increased activity of succinyl-CoA synthetase (SCS and oxalate CoA-transferase (OCT in the Al-stressed cells afforded an effective route to ATP synthesis from oxalyl-CoA via substrate level phosphorylation. This modified TCA cycle with diminished efficacy in NADH production and decreased CO(2-evolving capacity, orchestrates the synthesis of oxalate, NADPH, and ATP, ingredients pivotal to the survival of P. fluorescens in an Al environment. The channeling of succinyl-CoA towards ATP formation may be an important function of the TCA cycle during anaerobiosis, Fe starvation and O(2-limited conditions.

  3. ATP-Bioluminescence as a method to evaluated microbiological quality of UHT milk

    Directory of Open Access Journals (Sweden)

    A.F. Cunha

    2014-12-01

    Full Text Available New approaches are needed to quickly indicate possible contamination of UHT milk, among them the technique of ATP-Bioluminescence. Therefore, the aim of this study was to compare the results of culture methods with the results of ATP-Bioluminescence technique of 102 UHT whole milk samples incubated at 48, 72, and 168 hours. UHT milk samples were analyzed for the presence of mesophilic and psychrotrophic aerobic microorganisms using Plate Count Agar (PCA, Brain-Heart Infusion (BHI media and PetrifilmTM Aerobic Count (AC plates. The ATP-Bioluminescence technique was applied through the Microbial Luminescent Screening (MLS system. Significant correlations were found between counts of aerobic mesophilic microorganisms on PCA, PetrifilmTM AC, BHI and results of ATP bioluminescence technique (P≤0.05. The ATP-Bioluminescence technique had higher correlation with counting method in PCA than BHI media. At lower pass/fail limits of Relative Light Units (60, 50, 45 and 40 RLU, the number of samples identified as positive increased and statistically agreed with aerobic mesophilic microorganism counts (P>0.05. For the dairy industry, the ATP-Bioluminescence technique may become an important tool that assists the official methods to quickly monitor the microbiological quality of UHT milk though this will likely require a threshold below 150 RLU.

  4. ATP4 and ciliation in the neuroectoderm and endoderm of Xenopus embryos and tadpoles

    Directory of Open Access Journals (Sweden)

    Peter Walentek

    2015-09-01

    Full Text Available During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/β-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012 516–527. and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015 of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/β-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/β-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.

  5. Design, synthesis and evaluation of redox radiopharmaceuticals: a potential new approach for the development of brain imaging agents

    International Nuclear Information System (INIS)

    The fabrication and complete evaluation are described of a dihydropyridine in equilibrium pyridinium salt type redox system for the delivery of radioiodinated agents to the brain. The pivotal intermediate, N-succinimidyl (1-methylpyridinium iodide)-3-carboxylate was prepared by condensation of nicotinic acid and N-hydroxysuccinimide in the presence of dicyclohexylcarbodimide, followed by quaternization of III with methyl iodide. Tissue distribution studies of 125I-labeled 4-iodoaniline and the redox agents were performed in rats. [125I]Iodoaniline initially showed moderate (0.58% dose/gm) brain uptake with subsequent release of the radioactivity from the brain. [125I]Iodoaniline, when coupled to a dihydropyridine carrier showed higher uptake and retention in the brain. The [125I]iodophenylethyl analogue showed uptake and retention in the brain to be very similar. Apparently the lipophilic agents cross the blood-brain barrier and are oxidized (quaternized) within the brain. The blood-brain barrier then prevents their release resulting in high uptake and retention in the brain and high brain:blood ratios. 11 refs., 3 figs

  6. A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake

    OpenAIRE

    Hicks, David B.; Wang, Zhenxiong; Wei, Yi; Kent, Rebecca; Guffanti, Arthur A.; Banciu, Horia; Bechhofer, David H.; Krulwich, Terry A.

    2003-01-01

    The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin ...

  7. The structural basis of ATP as an allosteric modulator.

    OpenAIRE

    Shaoyong Lu; Wenkang Huang; Qi Wang; Qiancheng Shen; Shuai Li; Ruth Nussinov; Jian Zhang

    2014-01-01

    Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP...

  8. The Structural Basis of ATP as an Allosteric Modulator

    OpenAIRE

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-01-01

    Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP...

  9. ATP responses in human C nociceptors.

    Science.gov (United States)

    Hilliges, Marita; Weidner, Christian; Schmelz, Martin; Schmidt, Roland; Ørstavik, Kristin; Torebjörk, Erik; Handwerker, Hermann

    2002-07-01

    Microelectrode recordings of impulse activity in nociceptive C fibres were performed in cutaneous fascicles of the peroneal nerve at the knee level in healthy human subjects. Mechano-heat responsive C units (CMH), mechano-insensitive but heat-responsive (CH) as well as mechano-insensitive and heat-insensitive C units (CM(i)H(i)) were identified. A subgroup of the mechano-insensitive units was readily activated by histamine. We studied the responsiveness of these nociceptor classes to injection of 20 microl 5 mM adenosintriphosphate (ATP) using saline injections as control. Because of mechanical distension during injection, which typically activates mechano-responsive C fibres, interest was focused on responsiveness to ATP after withdrawal of the injection needle. Post-injection responses were observed in 17/27 (63%) mechano-responsive units and in 14/22 (64%) mechano-insensitive units. Excitation by ATP occurred in 9/11 CH units and in 5/11 CM(i)H(i) units. ATP responsive units were found both within the histamine-responsive and the histamine-insensitive group of mechano-insensitive fibres. ATP responses appeared with a delay of 0-180 s after completion of injection; responses were most pronounced during the first 1-3 min of activation, and irregular ongoing activity was observed for up to 10 or even 20 min. ATP responses were dose-dependent, concentrations lower than 5 mM gave weaker responses. No heat or mechanical sensitisation was observed in any of the major fibre classes. In conclusion, we have shown that ATP injections at high concentrations activate C-nociceptors in healthy human skin, without preference for mechano-responsive or mechano-insensitive units. ATP did not sensitise human C fibres for mechanical or heat stimuli. We discuss how various mechanisms might contribute to the observed responses to ATP. PMID:12098617

  10. Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier

    DEFF Research Database (Denmark)

    Thomsen, Maj Schneider; Birkelund, Svend; Burkhart, Annette;

    2016-01-01

    The brain vascular basement membrane is important for both blood-brain barrier (BBB) development, stability, and barrier integrity and the contribution hereto from brain capillary endothelial cells (BCECs), pericytes, and astrocytes of the BBB is probably significant. The aim of the present study......-culture, in co-culture with pericytes or mixed glial cells, or as a triple-culture with both pericytes and mixed glial cells. The integrity of the BBB models was validated by measures of transendothelial electrical resistance (TEER) and passive permeability to mannitol. The expression of basement membrane...... proteins was analysed using RT-qPCR, mass spectrometry, and immunocytochemistry. Co-culturing mBCECs with pericytes, mixed glial cells, or both significantly increased the TEER compared to the mono-culture, and a low passive permeability was correlated with high TEER. The mBCECs expressed all major...

  11. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory & urological disorders

    Directory of Open Access Journals (Sweden)

    Anthony Ford

    2013-12-01

    Full Text Available A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates & sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X & P2Y receptors mediate ATP modulation of sensory pathways & participate in dysregulation, where ATP action directly on primary afferent neurons (PANs, linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice & knock-down in rats led to reduced nocifensive activity & visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory & visceral pain models, have emerged. Significantly, these compounds have no overt CNS action & are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral & central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral “hollow” organs primes them to chronic discomfort, irritation & pain (symptoms as well as exacerbated autonomic reflexes (signs, & how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary & airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional & sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs & symptoms, in the potential for benefit of P2X3 antagonists.

  12. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures.

    Science.gov (United States)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G; Møller, Lisbeth B

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  13. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    OpenAIRE

    Jun Adachi; Marina Kishida; Shio Watanabe; Yuuki Hashimoto; Kazuna Fukamizu; Takeshi Tomonaga

    2015-01-01

    Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified ...

  14. ATP differentially upregulates fibroblast growth factor 2 and transforming growth factor α in neonatal and adult mice: effect on neuroproliferation.

    Science.gov (United States)

    Jia, C; Cussen, A R; Hegg, C C

    2011-03-17

    Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development. PMID:21187124

  15. ATP differentially upregulates growth factors FGF2 and TGFα in neonatal and adult mice: Effect on neuroproliferation

    Science.gov (United States)

    Jia, Cuihong; Cussen, Amber R.; Hegg, Colleen Cosgrove

    2011-01-01

    Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium; however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development. PMID:21187124

  16. Synthesis of some 11C-labelled MAO-A inhibitors and their in vivo uptake kinetics in rhesus monkey brain

    International Nuclear Information System (INIS)

    Five potential MAO-A inhibitors--harmine, N-methyl-harmine, harmaline, brofaromine, and clorgyline--were labelled with 11C and their brain kinetics evaluated in vivo in rhesus monkey using PET. The compounds were synthesized by alkylation with 11C methyl iodide and obtained in 48-89% radiochemical yield within 40 to 45 min synthesis time and with specific radioactivities in the region of 0.49-2.4 Ci μmol-1 (18-87 GBq μmol-1) at the end of synthesis. The kinetic pattern after administration of MAO-A inhibitors was comparable to that seen in the tracer study when using 11C-brofaromine, 11C-harmaline, or 11C-clorgyline, although the magnitude of uptake markedly increased in the case of brofaromine and harmaline. Both 11C-methylharmine and 11C-harmine showed a significant washout in the inhibition studies. The kinetics of brain uptake with and without MAO-A inhibition is compatible with a significant fraction of the tracer bound to MAO-A for 11C-methylharmine and 11C-harmine, whereas 11C-brofaromine, 11C-harmaline, or 11C-clorgyline did not seem to show specific enzyme binding

  17. Effects of Phellinus linteus administration on serotonin synthesis in the brain and expression of monocarboxylate transporters in the muscle during exhaustive exercise in rats.

    Science.gov (United States)

    Seo, Jin-Hee; Sung, Yun-Hee; Kim, Ki-Jeong; Shin, Mal-Soon; Lee, Eun-Kyu; Kim, Chang-Ju

    2011-01-01

    This study was conducted to determine the effects of Phellinus linteus (PL) on serotonin synthesis in the brain and on the expression of monocarboxylate transporters (MCTs) in muscles during exhaustive exercise in rats. In this study, 60 male Sprague-Dawley rats were divided into the following 6 groups: control; exercise; exercise and 50 mg/kg of PL treatment; exercise and 100 of mg/kg PL treatment; exercise and 200 mg/kg of PL treatment; and exercise and 100 mg/kg of caffeine treatment. Treatment with 200 mg/kg of PL led to a significant increase in the time to exhaustion in response to running on a treadmill and a significant decrease in 5-hydroxytryptamine synthesis and tryptophan hydroxylase expression in the dorsal raphe of rats. MCT1 and MCT4 expression of the gastrocnemius muscles was also increased in response to treatment with 200 mg/kg of PL. The results of the present study demonstrated that the administration of PL increased endurance exercise performance through inhibition of serotonin production in the brain and increased the expression of MCT1 and MCT4 in muscles. These results suggest that PL exerts an ergogenic effect. PMID:21512297

  18. Positive Inotropic Effects of Low dATP/ATP Ratios on Mechanics and Kinetics of Porcine Cardiac Muscle

    OpenAIRE

    Schoffstall, Brenda; Clark, Amanda; Chase, P. Bryant

    2006-01-01

    Substitution of 2′-deoxy ATP (dATP) for ATP as substrate for actomyosin results in significant enhancement of in vitro parameters of cardiac contraction. To determine the minimal ratio of dATP/ATP (constant total NTP) that significantly enhances cardiac contractility and obtain greater understanding of how dATP substitution results in contractile enhancement, we varied dATP/ATP ratio in porcine cardiac muscle preparations. At maximum Ca2+ (pCa 4.5), isometric force increased linearly with dAT...

  19. The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH)

    International Nuclear Information System (INIS)

    Introduction: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo. We report here the synthesis and in vivo evaluation of [18F]PF-9811, a novel PET ligand for non-invasive imaging of FAAH in the brain. Methods: The potency and selectivity of unlabeled PF-9811 were determined by activity-based protein profiling (ABPP) both in vitro and in vivo. [18F]PF-9811 was synthesized in a 3-step, one-pot reaction sequence, followed by HPLC purification. Biological evaluation was performed by biodistribution and dynamic PET imaging studies in male rats. The specificity of [18F]PF-9811 uptake was evaluated by pre-administration of PF-04457845, a potent and selective FAAH inhibitor, 1 h prior to radiotracer injection. Results: Biodistribution studies show good uptake (SUV ∼ 0.8 at 90 min) of [18F]PF-9811 in rat brain, with significant reduction of the radiotracer in all brain regions (37%–73% at 90 min) in blocking experiments. Dynamic PET imaging experiments in rat confirmed the heterogeneous uptake of [18F]PF-9811 in brain regions with high FAAH enzymatic activity, as well as statistically significant reductions in signal following pre-administration of the blocking compound PF-04457845. Conclusions: [18F]PF-9811 is a promising PET imaging agent for FAAH. Biodistribution and PET imaging experiments show that the tracer has good uptake in brain, regional heterogeneity, and specific binding as determined by blocking experiments with the highly potent and selective FAAH inhibitor, PF-04457845.

  20. ATP-dependent protease in maize mitochondria

    International Nuclear Information System (INIS)

    ATP-dependent protease was identified in the matrix of Zea mays L. Sachara mitochondria. 14C-methylated casein has been used as a substrate, and the matrix ATP-dependent protease exhibited similar sensitivity towards specific inhibitors as the Lon protease from E. coli nd analogues from rat liver and yeast mitochondria. Here we report the existence of Lon like ATP-dependent protease in intact mitochondria prepared from 4-days-old epicotyls of Zea mays L. seedling. Enzyme has been purified from Lubrol treated mitochondria using ion exchange chromatography and gel filtration. The enzyme activity has been estimated using 14C-methylated casein as a substrate and sensitivity of the protease towards the specific inhibitors has been tested. ATP-dependent protease from the mitochondrial matrix of maize exhibit similar sensitivity to the above mentioned inhibitors like Lon protease from yeast and rat liver mitochondria as well as from E. coli. (authors)

  1. Customized ATP towpreg. [Automated Tow Placement

    Science.gov (United States)

    Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

    1992-01-01

    Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placement from the robot head to the tool. This study examined the key towpreg ribbon properties and dimensions which play a significant role in ATP. Analysis of the heat transfer process window indicates that adequate heating can be achieved at lay down rates as high as 1 m/sec. While heat transfer did not appear to be the limiting factor, resin flow and fiber movement into tow lap gaps could be. Accordingly, consideration was given to towpreg ribbon having uniform yet non-rectangular cross sections. Dimensional integrity of the towpreg ribbon combined with customized ribbon architecture offer great promise for processing advances in ATP of high performance composites.

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

    DEFF Research Database (Denmark)

    Mortensen, Stefan P.; Gonzalez-Alonso, Jose; Nielsen, Jens Jung;

    2009-01-01

    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 mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial...... ATP and 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 norepinephrine (NE) concentrations were measured during: 1) femoral arterial ATP infusion (0.......42+/-0.04 and 2.26+/-0.52 mumol/min; mean+/-SEM) and 2) one-leg knee-extensor exercise (18+/-0 and 37+/-2W) in 10 healthy, male subjects. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from ~0.3 L/min at baseline to 4.2+/-0.3 and 4.6+/-0.5 L/min, respectively, whereas...

  3. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    International Nuclear Information System (INIS)

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca2+ as the activating cation

  4. NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATP

    OpenAIRE

    Pelzmann, Brigitte; Hallström, Seth; Schaffer, Peter; Lang, Petra; Nadlinger, Karl; Birkmayer, George D; Vrecko, Karoline; Reibnegger, Gilbert; Koidl, Bernd

    2003-01-01

    The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured.Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36–37°C. Adenine nucleotid...

  5. [{sup 11}C]SMe-ADAM, an imaging agent for the brain serotonin transporter: synthesis, pharmacological characterization and microPET studies in rats

    Energy Technology Data Exchange (ETDEWEB)

    Zessin, Joerg [Institut fuer Bioanorganische und Radiopharmazeutische Chemie, Forschungszentrum Rossendorf, 01314 Dresden (Germany)]. E-mail: j.zessin@fz-rossendorf.de; Deuther-Conrad, Winnie [Institut fuer Interdisziplinaere Isotopenforschung, 04318 Leipzig (Germany); Kretzschmar, Marion [Institut fuer Bioanorganische und Radiopharmazeutische Chemie, Forschungszentrum Rossendorf, 01314 Dresden (Germany); Wuest, Frank [Institut fuer Bioanorganische und Radiopharmazeutische Chemie, Forschungszentrum Rossendorf, 01314 Dresden (Germany); Pawelke, Beate [Institut fuer Bioanorganische und Radiopharmazeutische Chemie, Forschungszentrum Rossendorf, 01314 Dresden (Germany); Brust, Peter [Institut fuer Interdisziplinaere Isotopenforschung, 04318 Leipzig (Germany); Steinbach, Joerg [Institut fuer Interdisziplinaere Isotopenforschung, 04318 Leipzig (Germany); Bergmann, Ralf [Institut fuer Bioanorganische und Radiopharmazeutische Chemie, Forschungszentrum Rossendorf, 01314 Dresden (Germany)

    2006-01-15

    N,N-Dimethyl-2-(2-amino-4-methylthiophenylthio)benzylamine (S Me-Adam, 1) is a highly potent and selective inhibitor of the serotonin transporter (SPERT). This compound was labeled with carbon-11 by methylation of the S-desmethyl precursor 10 with [{sup 11}C]methyl iodide to obtain the potential positron emission tomography (PET) radioligand [{sup 11}C]S Me-Adam. The radiochemical yield was 27{+-}5%, and the specific radioactivity was 26-40 GBq/{mu}mol at the end of synthesis. Ex vivo and in vivo biodistribution experiments in rats demonstrated a rapid accumulation of the radiotracer in brain regions known to be rich in SPERT, such as the thalamus/hypothalamus region (3.59{+-}0.41%ID/g at 5 min after injection). The specific uptake reached a thalamus to cerebellum ratio of 6.74{+-}0.95 at 60 min postinjection. The [{sup 11}C]SMe-ADAM uptake in the thalamus was significantly decreased by pretreatment with fluoxetine to 38{+-}11% of the control value. Furthermore, no metabolites of [{sup 11}C]SMe-ADAM could be detected in the SERT-rich regions of the rat brain. It is concluded that [{sup 11}C]SMe-ADAM may be a suitable PET ligand for SERT imaging in the living brain.

  6. [11C]SMe-ADAM, an imaging agent for the brain serotonin transporter: synthesis, pharmacological characterization and microPET studies in rats

    International Nuclear Information System (INIS)

    N,N-Dimethyl-2-(2-amino-4-methylthiophenylthio)benzylamine (S Me-Adam, 1) is a highly potent and selective inhibitor of the serotonin transporter (SPERT). This compound was labeled with carbon-11 by methylation of the S-desmethyl precursor 10 with [11C]methyl iodide to obtain the potential positron emission tomography (PET) radioligand [11C]S Me-Adam. The radiochemical yield was 27±5%, and the specific radioactivity was 26-40 GBq/μmol at the end of synthesis. Ex vivo and in vivo biodistribution experiments in rats demonstrated a rapid accumulation of the radiotracer in brain regions known to be rich in SPERT, such as the thalamus/hypothalamus region (3.59±0.41%ID/g at 5 min after injection). The specific uptake reached a thalamus to cerebellum ratio of 6.74±0.95 at 60 min postinjection. The [11C]SMe-ADAM uptake in the thalamus was significantly decreased by pretreatment with fluoxetine to 38±11% of the control value. Furthermore, no metabolites of [11C]SMe-ADAM could be detected in the SERT-rich regions of the rat brain. It is concluded that [11C]SMe-ADAM may be a suitable PET ligand for SERT imaging in the living brain

  7. Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

    Energy Technology Data Exchange (ETDEWEB)

    Rhodes, C.J.; Lucas, C.A.; Mutkoski, R.L.; Orci, L.; Halban, P.A.

    1987-08-05

    Isolated rat pancreatic islets were pulse-labeled for 5 min with (/sup 3/H)leucine then chased for 25 min, during which time endogenously labeled (/sup 3/H)proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled (/sup 3/H)proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer, proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect.

  8. Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

    International Nuclear Information System (INIS)

    Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer, proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect

  9. ATP as a signaling molecule: the exocrine focus

    DEFF Research Database (Denmark)

    Novak, Ivana

    2003-01-01

    Why and how do cells release ATP? It is not spilled energy. ATP becomes an extracellular regulator. Various cellular responses are initiated by purinergic receptors and signaling processes and are terminated by breakdown of ATP by ectonucleotidases. In epithelia, ATP regulates salt and water...

  10. Pigment epithelium-derived factor binds to cell-surface F(1)-ATP synthase.

    Science.gov (United States)

    Notari, Luigi; Arakaki, Naokatu; Mueller, David; Meier, Scott; Amaral, Juan; Becerra, S P

    2010-05-01

    Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a approximately 60 kDa PEDF-binding protein in bovine retina with Bos taurus F(1)-ATP synthase beta-subunit, and that F(1)F(o)-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F(1). Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F(1). Real-time binding as determined by surface plasmon resonance demonstrated that yeast F(1) interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F(1) and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F(1). Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of approximately 60 kDa. Antibodies to F(1)beta-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F(1), these results show that PEDF is a ligand for endothelial cell-surface F(1)F(o)-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. PMID:20412062

  11. Resonance phenomenon of the ATP motor as an ultrasensitive biosensor.

    Science.gov (United States)

    Wang, Peirong; Zhang, Xiaoguang; Zhang, Xu; Wang, Xia; Li, Xueren; Yue, Jiachang

    2012-09-28

    We designed a rotary biosensor as a damping effector, with the rotation of the F(0)F(1)-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection. PMID:22960174

  12. Design, Synthesis and Biological Evaluation of Brain-Targeted Thiamine Disulfide Prodrugs of Ampakine Compound LCX001

    OpenAIRE

    Dian Xiao; Fan-Hua Meng; Wei Dai; Zheng Yong; Jin-Qiu Liu; Xin-Bo Zhou; Song Li

    2016-01-01

    Ampakine compounds have been shown to reverse opiate-induced respiratory depression by activation of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors. However, their pharmacological exploitations are hindered by low blood-brain barrier (BBB) permeability and limited brain distribution. Here, we explored whether thiamine disulfide prodrugs with the ability of “lock-in” can be used to solve these problems. A series of thiamine disulfide prodrugs 7a–7f of ampakine co...

  13. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P-1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and C

  14. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU)

    Science.gov (United States)

    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases. PMID:27196432

  15. Physiological levels of ATP Negatively Regulate Proteasome Function

    OpenAIRE

    Huang, Hongbiao; Zhang, Xiaoyan; Li, Shujue; Liu, Ningning; Lian, Wen; McDowell, Emily; Zhou, Ping; Zhao, Canguo; Guo, Haiping; Zhang, Change; Yang, Changshan; Wen, Guangmei; Dong, Xiaoxian; Lu, Li; Ma, Ningfang

    2010-01-01

    Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 μM. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolyti...

  16. Release of endogenous ATP during sympathetic nerve stimulation.

    OpenAIRE

    Lew, M. J.; White, T. D.

    1987-01-01

    1 Vas deferens from guinea-pig was stimulated with a suction electrode and both contractions and release of endogenous ATP monitored 2 Release of ATP was tetrodotoxin-sensitive and increased when the number of stimuli was increased. 3 Release of ATP was not due to contraction of the muscle and persisted following block of contractions with prazosin and alpha, beta-methylene ATP. 4 These results indicate that stimulation of the sympathetic nerves in the vas deferens releases endogenous ATP pre...

  17. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

    Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

  18. Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena

    ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and ...... release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological......, particularly during Ca2+ stress conditions. In conclusion, these studies demonstrate a complex regulation of purinergic signalling in exocrine pancreas. A crucial role for duct cells in mediating extracellular nucleotides homeostasis, involving ATP release, subsequent hydrolysis and conversion via...

  19. Bedaquiline – The first ATP synthase inhibitor against multi drug resistant tuberculosis

    OpenAIRE

    Lakshmanan, Mageshwaran; Xavier, Alphienes Stanley

    2013-01-01

    Increasing incidence of MDR-TB, long duration of treatment and co-infection with HIV are the significant problems in achieving the eradication of tuberculosis. Bedaquiline is an anti-tuberculosis drug with unique mechanism of action. It selectively inhibits the mycobacterial energy metabolism i.e. ATP synthesis and found to be effective against all states of Mycobacterium tuberculosis like active, dormant, replicating, non-replicating, intracellular and extracellular. Preclinical studies have...

  20. Ibogaine labeling with 99mTc-tricarbonyl: synthesis and transport at the mouse blood-brain barrier.

    Science.gov (United States)

    Tournier, Nicolas; André, Pascal; Blondeel, Sandy; Rizzo-Padoin, Nathalie; du Moulinet d'Hardemarre, Amaury; Declèves, Xavier; Scherrmann, Jean-Michel; Cisternino, Salvatore

    2009-12-01

    The (99m)Tc-tricarbonyl core may be used as an ideal tool for gamma-labeling ligands in noninvasive SPECT imaging. However, most (99m)Tc-tricarbonyl-labeled agents have difficulty crossing the blood-brain barrier (BBB). We radiolabeled the neuroactive indole ibogaine with (99m)Tc-tricarbonyl and measured its transport into the mouse brain by in situ brain perfusion. We measured the interactions of [(99m)Tc(CO)(3)-ibogaine](+) and (99m)Tc-tricarbonyl with the main BBB efflux transporters P-gp and BCRP in vitro and in vivo. Ibogaine was radiolabeled (yield: over 95%). [(99m)Tc(CO)(3)-ibogaine](+) entered the brain (K(in)) poorly (0.18 microL/g/s), at about the same rate as (99m)Tc-tricarbonyl (0.16 microL/g/s) and [(99m)Tc-sestamibi](+) (0.10 microL/g/s). The CNS tracer [(99m)Tc-HMPAO](0) entered the brain approximately 70-times higher than [(99m)Tc(CO)(3)-ibogaine](+). In vitro studies revealed that neither [(99m)Tc(CO)(3)-ibogaine](+) nor (99m)Tc-tricarbonyl ion were substrates for P-gp or BCRP. But lowering the membrane dipole potential barrier with phloretin enhanced the brain transport of [(99m)Tc(OH(2))(3)(CO)(3)](+) approximately 3-fold. Thus, ibogaine directly labeled with (99m)Tc-tricarbonyl is not suitable for CNS imaging because of its poor uptake. Brain transport is not restricted by efflux transporters but is reduced by its lipophilicity and interaction with the membrane-positive dipole potential. PMID:19492342

  1. Open State Destabilization by Atp Occupancy Is Mechanism Speeding Burst Exit Underlying KATP Channel Inhibition by Atp

    OpenAIRE

    Li, Lehong; Geng, Xuehui; Drain, Peter

    2002-01-01

    The ATP-sensitive potassium (KATP) channel is named after its characteristic inhibition by intracellular ATP. The inhibition is a centerpiece of how the KATP channel sets electrical signaling to the energy state of the cell. In the β cell of the endocrine pancreas, for example, ATP inhibition results from high blood glucose levels and turns on electrical activity leading to insulin release. The underlying gating mechanism (ATP inhibition gating) includes ATP stabilization of closed states, bu...

  2. Real time response of brain phosphocreatine to a 4S workload determined by 31P fMRS

    International Nuclear Information System (INIS)

    Full text: Phosphocreatine (PCr) is suggested to play a major role in buffering ATP in the brain and, as such, upon induction of mental work, PCr is initially the major source of high-energy phosphate bonds. We have developed a real-time functional magnetic resonance spectroscopy (fMRS) protocol using the 31P nucleus which enables us to measure levels of high-energy phosphate compounds (ATP and PCr), brain pH and inorganic phosphate in response to an intense mental workload (4s of 8Hz flashing chequerboard stimulation). Using this paradigm we have obtained data with 0.5 s time resolution. This improves upon previous published time resolution of 3.5 s. The new paradigm shows that PCr levels decrease immediately following stimulus onset with levels recovering rapidly following stimulus cessation. This recovery results in an overshoot of resting levels before rebounding slightly and recovering to back resting levels ∼ 16s after cessation of stimulus. In this respect, the timecourse of PCr consumption and resupply mirrors those of glucose and oxygen under conditions of transient oxygen and glucose debt. During the same period cerebral pH increases linearly from resting levels. This is consistent with continual sequestration of H+ by ADP, reflecting synthesis of ATP. This suggests that the energy debt resulting from the workload is ongoing for some time following cessation of stimulus. Copyright (2002) Australian Neuroscience Society

  3. GMP-compliant automated synthesis of [18F]AV-45 (Florbetapir F 18) for imaging β-amyloid plaques in human brain

    International Nuclear Information System (INIS)

    We report herein the Good Manufacturing Practice (GMP)-compliant automated synthesis of 18F-labeled styrylpyridine, AV-45 (Florbetapir), a novel tracer for positron emission tomography (PET) imaging of β-amyloid (Aβ) plaques in the brain of Alzheimer's disease patients. [18F]AV-45 was prepared in 105 min using a tosylate precursor with Sumitomo modules for radiosynthesis under GMP-compliant conditions. The overall yield was 25.4±7.7% with a final radiochemical purity of 95.3±2.2% (n=19). The specific activity of [18F]AV-45 reached as high as 470±135 TBq/mmol (n=19). The present studies show that [18F]AV-45 can be manufactured under GMP-compliant conditions and could be widely available for routine clinical use.

  4. Design, Synthesis and Biological Evaluation of Brain-Targeted Thiamine Disulfide Prodrugs of Ampakine Compound LCX001

    Directory of Open Access Journals (Sweden)

    Dian Xiao

    2016-04-01

    Full Text Available Ampakine compounds have been shown to reverse opiate-induced respiratory depression by activation of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA glutamate receptors. However, their pharmacological exploitations are hindered by low blood-brain barrier (BBB permeability and limited brain distribution. Here, we explored whether thiamine disulfide prodrugs with the ability of “lock-in” can be used to solve these problems. A series of thiamine disulfide prodrugs 7a–7f of ampakine compound LCX001 was synthesized and evaluated. The trials in vitro showed that prodrugs 7e, 7d, 7f possessed a certain stability in plasma and quickly decomposed in brain homogenate by the disulfide reductase. In vivo, prodrug 7e decreased the peripheral distribution of LCX001 and significantly increased brain distribution of LCX001 after i.v. administration. This compound showed 2.23- and 3.29-fold greater increases in the AUC0-t and MRT0-t of LCX001 in brain, respectively, than did LCX001 itself. A preliminary pharmacodynamic study indicated that the required molar dose of prodrug 7e was only one eighth that of LCX001 required to achieve the same effect in mice. These findings provide an important reference to evaluate the clinical outlook of ampakine compounds.

  5. Nuclear genetic defects of mitochondrial ATP synthase

    Czech Academy of Sciences Publication Activity Database

    Hejzlarová, Kateřina; Mráček, Tomáš; Vrbacký, Marek; Kaplanová, Vilma; Karbanová, Vendula; Nůsková, Hana; Pecina, Petr; Houštěk, Josef

    2014-01-01

    Roč. 63, Suppl.1 (2014), S57-S71. ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/11/0970; GA ČR GAP303/12/1363; GA MZd(CZ) NT12370; GA MZd(CZ) NT14050 Grant ostatní: Univerzita Karlova(CZ) 370411 Institutional support: RVO:67985823 Keywords : mitochondrial diseases * TMEM70 * ATPAF1 * ATP5A1 * ATP5E Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.293, year: 2014

  6. Glucose modulates rat substantia nigra GABA release in vivo via ATP-sensitive potassium channels.

    OpenAIRE

    During, M J; Leone, P.; Davis, K. E.; Kerr, D; Sherwin, R S

    1995-01-01

    Glucose modulates beta cell insulin secretion via effects on ATP-sensitive potassium (KATP) channels. To test the hypothesis that glucose exerts a similar effect on neuronal function, local glucose availability was varied in awake rats using microdialysis in the substantia nigra, the brain region with the highest density of KATP channels. 10 mM glucose perfusion increased GABA release by 111 +/- 42%, whereas the sulfonylurea, glipizide, increased GABA release by 84 +/- 20%. In contrast, perfu...

  7. Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)*

    OpenAIRE

    Drozak, Jakub; Veiga-da-Cunha, Maria; Vertommen, Didier; Stroobant, Vincent; Van Schaftingen, Emile

    2010-01-01

    Carnosine (β-alanyl-l-histidine) and homocarnosine (γ-aminobutyryl-l-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 ...

  8. Calcium signals in the nucleus accumbens: Activation of astrocytes by ATP and succinate

    Directory of Open Access Journals (Sweden)

    Emri Zsuzsa

    2011-10-01

    Full Text Available Abstract Background Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc, and thereby to explore the action of citric acid cycle intermediate succinate (SUC. Results We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43 and the glial fibrillary acidic protein (GFAP indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthioadenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 μM in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. Conclusions We

  9. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

    Science.gov (United States)

    Yaniv, Yael; Spurgeon, Harold A.; Ziman, Bruce D.; Lyashkov, Alexey E.

    2013-01-01

    The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca2+-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca2+ cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca2+ (Ca2+m) and an indirect effect via enhanced Ca2+-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca2+ and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca2+m and cAMP increased concurrently with the increase in AP firing rate. When Ca2+m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca2+m and an increase in Ca2+ activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level. PMID:23604710

  10. Proteomic Analysis of Wild-type and Mutant Huntingtin-associated Proteins in Mouse Brains Identifies Unique Interactions and Involvement in Protein Synthesis*

    Science.gov (United States)

    Culver, Brady P.; Savas, Jeffrey N.; Park, Sung K.; Choi, Jeong H.; Zheng, Shuqiu; Zeitlin, Scott O.; Yates, John R.; Tanese, Naoko

    2012-01-01

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis. PMID:22556411

  11. Low brain magnesium in migraine

    International Nuclear Information System (INIS)

    Brain magnesium was measured in migraine patients and control subjects using in vivo 31-Phosphorus Nuclear Magnetic Resonance Spectroscopy. pMg and pH were calculated from the chemical shifts between Pi, PCr and ATP signals. Magnesium levels were low during a migraine attack without changes in pH. We hypothesize that low brain magnesium is an important factor in the mechanism of the migraine attack

  12. ATP regulates sodium channel kinetics in pancreatic islet beta cells

    OpenAIRE

    Zou, Na; Rupnik, Marjan

    2015-01-01

    Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP](i)) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP](i) remains unclear. Here, we demonstrated that increasing [ATP](i) within a certain range of concentrations (2-8 mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2 mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12 mM). F...

  13. Extracellular ATP signaling and homeostasis in plant cells

    OpenAIRE

    Sun, Jian; Zhang, Chunlan; Zhang, Xuan; Deng, Shurong; Zhao, Rui; Shen, Xin; Chen, Shaoliang

    2012-01-01

    Extracellular ATP (eATP) is now recognized as an important signaling agent in plant growth and defense response to environmental stimuli. eATP has dual functions in plant cell signaling, which is largely dependent on its concentration in the extracellular matrix (ECM). A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development. Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase) help control the eATP concentr...

  14. Synthesis and evaluation of ether containing {sup 99m}Tc-nitrido dithiocarbamate complexes as brain perfusion imaging agent

    Energy Technology Data Exchange (ETDEWEB)

    Mallia, Madhava B. [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Mathur, Anupam [Medical and Biological Products Program, Board of Radiation and Isotope Technology, Mumbai 400094 (India); Subramanian, Suresh [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Banerjee, Sharmila [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kothari, Kanchan [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Koiry, S.P. [TechnicalPhysics and Prototype Engineering Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Sarma, H.D. [Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Venkatesh, Meera [Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085 (India) and Medical and Biological Products Program, Board of Radiation and Isotope Technology, Mumbai 400094 (India)]. E-mail: meerav@apsara.barc.ernet.in

    2006-03-15

    In the present study, a series of {sup 99m}Tc-nitrido dithiocarbamate complexes containing ether linkages have been prepared and their brain perfusion characteristics studied. Two primary dithiocarbamates and two secondary dithiocarbamates were synthesized in >80% yield and were characterized by elemental analyses. The ligands were then labeled using a {sup 99m}Tc-nitrido intermediate, prepared from sodium pertechnetate using commercially available nitrido kit-vials, at a low ligand concentration of 0.1 mg. The prepared complexes were obtained in more than 95% yield and were characterized by paper electrophoresis and HPLC. All the complexes were found to be neutral and eluted out as a single species in HPLC. Biodistribution studies were carried out in normal Swiss mice. All the complexes showed uptake in the brain. {sup 99m}TcN complexes of secondary dithiocarbamates showed higher initial brain uptake (5 min p.i.) than their primary amine counterparts. However, all the complexes exhibited rapid washout from the brain.

  15. Synthesis and evaluation of ether containing 99mTc-nitrido dithiocarbamate complexes as brain perfusion imaging agent

    International Nuclear Information System (INIS)

    In the present study, a series of 99mTc-nitrido dithiocarbamate complexes containing ether linkages have been prepared and their brain perfusion characteristics studied. Two primary dithiocarbamates and two secondary dithiocarbamates were synthesized in >80% yield and were characterized by elemental analyses. The ligands were then labeled using a 99mTc-nitrido intermediate, prepared from sodium pertechnetate using commercially available nitrido kit-vials, at a low ligand concentration of 0.1 mg. The prepared complexes were obtained in more than 95% yield and were characterized by paper electrophoresis and HPLC. All the complexes were found to be neutral and eluted out as a single species in HPLC. Biodistribution studies were carried out in normal Swiss mice. All the complexes showed uptake in the brain. 99mTcN complexes of secondary dithiocarbamates showed higher initial brain uptake (5 min p.i.) than their primary amine counterparts. However, all the complexes exhibited rapid washout from the brain

  16. GLUCOCORTICOIDS REGULATE THE SYNTHESIS OF GFAP IN INTACT AND ADRENALECTOMIZED RATS BUT DO NOT AFFECT ITS EXPRESSION FOLLOWING BRAIN INJURY

    Science.gov (United States)

    We examined the effects of corticosterone (CORT) on the amount of glial fibrillary acidic protein (GFAP) in INTACT, adrenalectomized (ADX) and brain-damaged rats. hort (5 days)- to long-term (4 months) CORT administration by injection, pellet implantation, or in the drinking wate...

  17. Calcium and ATP control multiple vital functions

    Science.gov (United States)

    Verkhratsky, Alexei

    2016-01-01

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca2+ concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca2+ concentration (which in all life forms is kept around 50–100 nM) forms the basis for a universal intracellular signalling system in which Ca2+ acts as a second messenger. Maintenance of transmembrane Ca2+ gradients, in turn, requires ATP-dependent Ca2+ transport, thus further emphasizing the inseparable links between these two substances. Ca2+ signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca2+ signalling relies on cell specific Ca2+ signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca2+ signalling toolkits lead to aberrant Ca2+ signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377728

  18. Calcium and ATP control multiple vital functions.

    Science.gov (United States)

    Petersen, Ole H; Verkhratsky, Alexei

    2016-08-01

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca(2+) concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca(2+) concentration (which in all life forms is kept around 50-100 nM) forms the basis for a universal intracellular signalling system in which Ca(2+) acts as a second messenger. Maintenance of transmembrane Ca(2+) gradients, in turn, requires ATP-dependent Ca(2+) transport, thus further emphasizing the inseparable links between these two substances. Ca(2+) signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca(2+) signalling relies on cell specific Ca(2+) signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca(2+) signalling toolkits lead to aberrant Ca(2+) signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377728

  19. Nuclear genetic defects of mitochondrial ATP synthase

    Czech Academy of Sciences Publication Activity Database

    Houštěk, Josef; Kmoch, S.; Mayr, J. A.; Sperl, W.; Zeman, J.

    Bari : University of Bari, 2008. L5.3-L5.3. [IUBMB Symposium S1. 22.06.2008-26.06.2008, Bari] R&D Projects: GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z50110509 Keywords : spr2 * mitochondrial disease * ATP synthase defects * nuclear mutation Subject RIV: EB - Genetics ; Molecular Biology

  20. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy

    OpenAIRE

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

    2014-01-01

    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.

  1. The ATP requiring step in assembly of M13 procoat protein into microsomes is related to preservation of transport competence of the precursor protein

    OpenAIRE

    Wiech, Hans; Sagstetter, Maria; Müller, Günter; Zimmermann, Richard

    1987-01-01

    M13 procoat protein is processed to transmembrane coat protein by dog pancreas microsomes after completion of synthesis and in the absence of the signal recognition particle (SRP)/docking protein system. ATP is required for fast and efficient processing of procoat protein by microsomes in a reticulocyte lysate. Requirement for ATP is also observed in the absence of ribosomes or docking protein. This indicates the existence of a unique assembly pathway for procoat protein into microsomes which...

  2. Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

    Directory of Open Access Journals (Sweden)

    Carina Henriksen

    Full Text Available Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4 encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by

  3. Synthesis, radiosynthesis and biological evaluation of 1, 4-dihydroquinoline derivatives as new carriers for specific brain delivery

    Energy Technology Data Exchange (ETDEWEB)

    Foucout, L.; Bohn, P.; Dupas, G.; Marsais, F.; Levacher, V. [Laboratoire de Chimie Organique Fine et Heterocyclique, UMR 6014, IRCOF, CNRS, Universite et INSA de Rouen, B.P. 08 F-76131, Mont- Saint-Aignan Cedex (France); Gourand, F.; Dhilly, M.; Barre, L. [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, CEA/DSV/I2BM/CI-NAPS UMR6232, Universite de Caen Basse Normandie, Caen (France); Bohn, P.; Costentin, J. [Laboratoire de Neuropharmacologie Experimentale associe au CNRS, FRE-2735, Faculte de Medecine et de pharmacie, Universite de Rouen, F-76000 (France); Abbas, A. [Inserm-EPHE-Universite de Caen Basse-Normandie, Unite U923, GIP Cyceron, CHU Cote de Nacre, Caen (France)

    2009-07-01

    In spite of numerous reports dealing with the use of 1, 4-dihydro-pyridines as carriers to deliver biological active compounds to the brain, this chemical delivery system (CDS) suffers from poor stability of the 1, 4-dihydropyridine derivatives towards oxidation and hydration reactions seriously limiting further investigations in vivo. In an attempt to overcome these limitations, we report herein the first biological evaluation of more stable annellated NADH models in the quinoline series as relevant neuro-active drug-carrier candidates. The radiolabeled 1, 4-dihydroquinoline [{sup 11}C]1a was prepared to be subsequently peripherally injected in rats. The injected animals were sacrificed and brains were collected. The radioactivity measured in rat brain indicated a rapid penetration of the carrier [{sup 11}C]1a into the CNS. HPLC analysis of brain homogenates showed that oxidation of [{sup 11}C]1a into the corresponding quinolinium salt [{sup 11}C]4a was completed in less than 5 min. An in vivo evaluation in mice is also reported to illustrate the potential of such 1, 4-dihydroquinoline derivatives to transport a neuro-active drug in the CNS. For this purpose, g-aminobutyric acid (GABA), well known to poorly cross the brain blood barrier (BBB) was connected to this 1, 4-dihydroquinoline-type carrier. After i.p. injection of 1, 4-dihydroquinoline-GABA derivative 1b in mice, a significant alteration of locomotor activity (LMA) was observed presumably resulting from an enhancement of central GABAergic activity. These encouraging results give strong evidence for the capacity of carrier-GABA derivative 1b to cross the BBB and exert a pharmacological effect on the CNS. This study paves the way for further progress in designing new redox chemical delivery systems. (authors)

  4. Synthesis, radiosynthesis and biological evaluation of 1, 4-dihydroquinoline derivatives as new carriers for specific brain delivery

    International Nuclear Information System (INIS)

    In spite of numerous reports dealing with the use of 1, 4-dihydro-pyridines as carriers to deliver biological active compounds to the brain, this chemical delivery system (CDS) suffers from poor stability of the 1, 4-dihydropyridine derivatives towards oxidation and hydration reactions seriously limiting further investigations in vivo. In an attempt to overcome these limitations, we report herein the first biological evaluation of more stable annellated NADH models in the quinoline series as relevant neuro-active drug-carrier candidates. The radiolabeled 1, 4-dihydroquinoline [11C]1a was prepared to be subsequently peripherally injected in rats. The injected animals were sacrificed and brains were collected. The radioactivity measured in rat brain indicated a rapid penetration of the carrier [11C]1a into the CNS. HPLC analysis of brain homogenates showed that oxidation of [11C]1a into the corresponding quinolinium salt [11C]4a was completed in less than 5 min. An in vivo evaluation in mice is also reported to illustrate the potential of such 1, 4-dihydroquinoline derivatives to transport a neuro-active drug in the CNS. For this purpose, g-aminobutyric acid (GABA), well known to poorly cross the brain blood barrier (BBB) was connected to this 1, 4-dihydroquinoline-type carrier. After i.p. injection of 1, 4-dihydroquinoline-GABA derivative 1b in mice, a significant alteration of locomotor activity (LMA) was observed presumably resulting from an enhancement of central GABAergic activity. These encouraging results give strong evidence for the capacity of carrier-GABA derivative 1b to cross the BBB and exert a pharmacological effect on the CNS. This study paves the way for further progress in designing new redox chemical delivery systems. (authors)

  5. Reduced basal ATP synthetic flux of skeletal muscle in patients with previous acromegaly.

    Directory of Open Access Journals (Sweden)

    Julia Szendroedi

    Full Text Available BACKGROUND: Impaired mitochondrial function and ectopic lipid deposition in skeletal muscle and liver have been linked to decreased insulin sensitivity. As growth hormone (GH excess can reduce insulin sensitivity, we examined the impact of previous acromegaly (AM on glucose metabolism, lipid storage and muscular ATP turnover. PARTICIPANTS AND METHODS: Seven AM (4f/3 m, age: 46+/-4 years, BMI: 28+/-1 kg/m(2 and healthy volunteers (CON: 3f/4 m, 43+/-4 years, 26+/-2 kg/m(2 matched for age and body mass underwent oral glucose testing for assessment of insulin sensitivity (OGIS and ss-cell function (adaptation index, ADAP. Whole body oxidative capacity was measured with indirect calorimetry and spiroergometry. Unidirectional ATP synthetic flux (fATP was assessed from (31P magnetic resonance spectroscopy (MRS of calf muscle. Lipid contents of tibialis anterior (IMCLt and soleus muscles (IMCLs and liver (HCL were measured with (1H MRS. RESULTS: Despite comparable GH, insulin-like growth factor-1 (IGF-I and insulin sensitivity, AM had approximately 85% lower ADAP (p<0.01 and approximately 21% reduced VO(2max (p<0.05. fATP was similarly approximately 25% lower in AM (p<0.05 and related positively to ADAP (r = 0.744, p<0.01, but negatively to BMI (r = -0.582, p<0.05. AM had approximately 3 fold higher HCL (p<0.05 while IMCLt and IMCLs did not differ between the groups. CONCLUSIONS: Humans with a history of acromegaly exhibit reduced insulin secretion, muscular ATP synthesis and oxidative capacity but elevated liver fat content. This suggests that alterations in ss-cell function and myocellular ATP production may persist despite normalization of GH secretion after successful treatment of acromegaly.

  6. Uncoupling of oxidative phosphorylation and ATP synthase reversal within the hyperthermic heart.

    Science.gov (United States)

    Power, Amelia; Pearson, Nicholas; Pham, Toan; Cheung, Carlos; Phillips, Anthony; Hickey, Anthony

    2014-09-01

    Heart failure is a common cause of death with hyperthermia, and the exact cause of hyperthermic heart failure appears elusive. We hypothesize that the energy supply (ATP) of the heart may become impaired due to increased inner-mitochondrial membrane permeability and inefficient oxidative phosphorylation (OXPHOS). Therefore, we assessed isolated working heart and mitochondrial function. Ex vivo working rat hearts were perfused between 37 and 43.5°C and showed break points in all functional parameters at ~40.5°C. Mitochondrial high-resolution respirometry coupled to fluorometry was employed to determine the effects of hyperthermia on OXPHOS and mitochondrial membrane potential (ΔΨ) in vitro using a comprehensive metabolic substrate complement with isolated mitochondria. Relative to 37 and 40°C, 43°C elevated Leak O2 flux and depressed OXPHOS O2 flux and ∆Ψ. Measurement of steady-state ATP production from mitochondria revealed decreased ATP synthesis capacity, and a negative steady-state P:O ratio at 43°C. This approach offers a more powerful analysis of the effects of temperature on OXPHOS that cannot be measured using simple measures such as the traditional respiratory control ratio (RCR) or P:O ratio, which, respectively, can only approach 1 or 0 with inner-membrane failure. At 40°C there was only a slight enhancement of the Leak O2 flux and this did not significantly affect ATP production rate. Therefore, during mild hyperthermia (40°C) there is no enhancement of ATP supply by mitochondria, to accompany increasing cardiac energy demands, while between this and critical hyperthermia (43°C), mitochondria become net consumers of ATP. This consumption may contribute to cardiac failure or permanent damage during severe hyperthermia. PMID:25263202

  7. Increased Intracellular [dATP] Enhances Cardiac Contraction in Embryonic Chick Cardiomyocytes

    OpenAIRE

    Schoffstall, Brenda; Chase, P. Bryant

    2008-01-01

    Although ATP is the physiological substrate for cardiac contraction, cardiac contractility is significantly enhanced in vitro when only 10% of ATP substrate is replaced with 2’-deoxy-ATP (dATP). To determine the functional effects of increased intracellular [dATP] ([dATP]i) within living cardiac cells, we used hypertonic loading with varying exogenous dATP/ATP ratios, but constant total nucleotide concentration, to elevate [dATP]i in contractile monolayers of embryonic chick cardiomyocytes. T...

  8. The effect of delta-aminolevulinic acid on the synthesis and metabolism of GABA in rabbit brain homogenates

    International Nuclear Information System (INIS)

    The porphyrin precursor delta-aminolevulinic acid (delta-ALA) is a structural analogue of the putative amino acid neurotransmitter, γ-aminobutyric acid (GABA). This study has demonstrated that delta-ALA has no effect on glutamate decarboxylase activity and only a small inhibitory effect on GABA aminotransferase activity. This would suggest that if accumulation of delta-ALA is related to development of the acute attack of porphyria, it is not via an effect on GABA synthesis and metabolism

  9. Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p

    OpenAIRE

    Rak, Malgorzata; Su, Chen Hsien; Xu, Jonathan Tong; Azpiroz, Ricardo; Singh, Angela Mohan; Tzagoloff, Alexander

    2016-01-01

    Expression of the mitochondrially encoded ATP6 and ATP8 genes is translationally regulated by F1 ATPase. We report a translational repressor (Smt1p) of the ATP6/8 mRNA that, when mutated, restores translation of the encoded Atp6p and Atp8p subunits of the ATP synthase. Heterozygous smt1 mutants fail to rescue the translation defect, indicating that the mutations are recessive. Smt1p is an intrinsic inner membrane protein, which, based on its sedimentation, has a native size twice that of the ...

  10. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa;

    2006-01-01

    -generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes...

  11. Reinterpreting the action of ATP analogs on K(ATP) channels.

    Science.gov (United States)

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

    2013-06-28

    Neuroendocrine-type K(ATP) 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 β-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'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]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 K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity. PMID:23665564

  12. Cloning of the cDNA for the human ATP synthase OSCP subunit (ATP5O) by exon trapping and mapping to chromosome 21q22.1-q22.2

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiming [Geneva Univ. Medical School (Switzerland); Morris, M.A.; Rossier, C. [Cantonal Hospital, Geneva (Switzerland)] [and others

    1995-08-10

    Exon trapping was used to clone portions of potential genes from human chromosome 21. One trapped sequence showed striking homology with the bovine and rat ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit. We subsequently cloned the full-length human ATP synthase OSCP cDNA (GDB/HGMW approved name ATP50) from infant brain and muscle libraries and determined its nucleotide and deduced amino acid sequence (EMBL/GenBank Accession No. X83218). The encoded polypeptide contains 213 amino acids, with more than 80% identity to bovine and murine ATPase OSCP subunits and over 35% identity to Saccharomyces cerevisiae and sweet potato sequences. The human ATP5O gene is located at 21q22.1-q22.2, just proximal to D21S17, in YACs 860G11 and 838C7 of the Chumakov et al. YAC contig. The gene is expressed in all human tissues examined, most strongly in muscle and heart. This ATP5O subunit is a key structural component of the stalk of the mitochondrial respiratory chain F{sub 1}F{sub 0}-ATP synthase and as such may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome (trisomy 21). 39 refs., 5 figs.

  13. Design, synthesis and preliminary bio-evaluation of glucose-cholesterol derivatives as ligands for brain targeting liposomes

    Institute of Scientific and Technical Information of China (English)

    Fan Lei; Wei Fan; Xian Kun Li; Shan Wang; Li Hai; Yong Wu

    2011-01-01

    A series of glucose-cholesterol derivatives 8a-8e as ligands for brain targeting liposomes were synthesized. The preparation of compound 6 involved temporary protection of glucose with chlorotrimethylsilicane and hexamethyldisilazane followed by selectively hydrolyzed. The known cholesteryl tosylate 1 were coupled to ethylene glycols to afford alcohol 2a-2e. Substitution and deprotection of alcohol 2a-2e furnished the acids 4a-4e, which was condensed with compound 6 to get compounds 7a-7e, and then was deprotected in tetrahydrofuran with TEA to obtain the title compounds. As a model drug, tegafur was entrapped by liposomes coupled with 8b, and preliminary in vivo evaluation shown 8b could enhance the ability of liposomes delivering tegafur across the blood brain barrier.

  14. Design, synthesis and evaluation of 2-deoxy-2-iodovinyl-branched carbohydrates as potential brain imaging agents

    International Nuclear Information System (INIS)

    Radioiodinated carbohydrates such as 2-deoxy-2-iodo-D-glucose and 3-deoxy-3-iodo-D-glucose undergo facile chemical or in vivo deiodination which precludes their use as radiotracers of glucose metabolism in tissues. To overcome the problems resulting from in vivo deiodination, we explored the concept of stabilizing radioiodide on a model carbohydrate, (E)-C-3-iodovinyl-D-allose (10) as an iodovinyl moiety. This agent did not exhibit brain specificity but showed low in vivo deiodination which demonstrated for the first time that radioiodide can be stabilized on a carbohydrate. The goal of this study was to develop a deoxy-branched carbohydrate with radioiodide stabilized as a vinyliodide with the objective of achieving high brain uptake. 10 refs., 1 fig., 1 tab

  15. Design, synthesis and evaluation of 2-deoxy-2-iodovinyl-branched carbohydrates as potential brain imaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Goodman, M.M.; Callahan, A.P.; Knapp, F.F. Jr.

    1986-01-01

    Radioiodinated carbohydrates such as 2-deoxy-2-iodo-D-glucose and 3-deoxy-3-iodo-D-glucose undergo facile chemical or in vivo deiodination which precludes their use as radiotracers of glucose metabolism in tissues. To overcome the problems resulting from in vivo deiodination, we explored the concept of stabilizing radioiodide on a model carbohydrate, (E)-C-3-iodovinyl-D-allose (10) as an iodovinyl moiety. This agent did not exhibit brain specificity but showed low in vivo deiodination which demonstrated for the first time that radioiodide can be stabilized on a carbohydrate. The goal of this study was to develop a deoxy-branched carbohydrate with radioiodide stabilized as a vinyliodide with the objective of achieving high brain uptake. 10 refs., 1 fig., 1 tab.

  16. The Role of ATP in the Regulation of NCAM Function

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a direct...... ATP-NCAM interaction and discuss its functional implications. The ectodomain of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP...... overlaps with the site of NCAM-FGFR interaction, and ATP is capable of disrupting NCAM-FGFR binding. This implies that NCAM signaling through FGFR can be regulated by ATP, which is supported by the observation that ATP can abrogate NCAM-induced neurite outgrowth. Finally, ATP can induce NCAM ectodomain...

  17. A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

    Science.gov (United States)

    Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

    2016-07-01

    In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. PMID:27154705

  18. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    Science.gov (United States)

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target. PMID:26621635

  19. The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance.

    Science.gov (United States)

    Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R; Junge, Wolfgang; Khan, Shahid

    2015-09-01

    The proton-driven ATP synthase (FOF1) is comprised of two rotary, stepping motors (FO and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may

  20. External Dentin Stimulation Induces ATP Release in Human Teeth.

    Science.gov (United States)

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain. PMID:26130258

  1. Comparative Features of Copper ATPases ATP7A and ATP7B Heterologously Expressed in COS-1 Cells

    OpenAIRE

    Liu, Yueyong; Pilankatta, Rajendra; Hatori, Yuta; Lewis, David; Inesi, Giuseppe

    2010-01-01

    ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, ...

  2. Synthesis and biodistribution of nitrido technetium-99m radiopharmaceuticals with dithiophosphinate ligands: a class of brain imaging agents

    International Nuclear Information System (INIS)

    The symmetrical complexes [99mTc][TcN(R2PS2)2] [R = CH3, CH2CH3, CH2CH2CH3, CH2(CH3)2], and the unsymmetrical complex [99mTc][TcN(Me2PS2)(Et2PS2)] have been prepared, at tracer level, through a two-step procedure involving the preliminary formation of a prereduced technetium nitrido intermediate followed by substitution reaction onto this species by the appropriate dithiophosphinate ligand [R2PS2]Na. The chemical identity of the resulting complexes have been established by comparison with the corresponding 99Tc-analogs prepared, at macroscopic level, by reacting the complex [99TcNCl4] [n-Bu4N] (n-Bu = n-butyl) with an excess of ligand in methanol, and characterized by elemental analyses and spectroscopic techniques. The complexes are neutral and lipophilic, and possess a square pyramidal geometry, with an apical Tc N group and two dithiophosphinate ligands spanning the four positions on the basal plane through the four sulfur atoms of the >PS2 group. In vitro studies showed that these radiopharmaceuticals are stable in solution and that their chemical identity was not altered after incubation with rat blood. Biodistribution studies have been carried out in rats and primates. The results demonstrate that these compounds are significantly retained into the brain of these animals for a prolonged time. Planar gamma camera images have been obtained in monkeys showing a good visualization of the cerebral region. However, the existence of persistent blood activity yields a brain/blood ratio lower than that observed with other 99mTc-based brain perfusion imaging agents

  3. Cellular proliferation and infiltration following interstitial irradiation of normal dog brain is altered by an inhibitor of polyamine synthesis

    International Nuclear Information System (INIS)

    Purpose: The objectives of this study were to quantitatively define proliferative and infiltrative cell responses after focal 125I irradiation of normal brain, and to determine the effects of an intravenous infusion of α-difluoromethylornithine (DFMO) on those responses. Methods and Materials: Adult beagle dogs were irradiated using high activity 125I sources. Saline (control) or DFMO (150 mg/kg/day) was infused for 18 days starting 2 days before irradiation. At varying times up to 8 weeks after irradiation, brain tissues were collected and the cell responses in and around the focal lesion were quantified. Immunohistochemical stains were used to label astrocytes (GFAP), vascular endothelial cells (Factor VIII), polymorphonuclear leukocytes (PMNs; MAC 387) and cells synthesizing deoxyribonucleic acid (DNA) (BrdU). Cellular responses were quantified using a histomorphometric analysis. Results: After radiation alone, cellular events included a substantial acute inflammatory response followed by increased BrdU labeling and progressive increases in numbers of capillaries and astrocytes. α-Difluoromethylornithine treatment significantly affected the measured cell responses. As in controls, an early inflammatory response was measured, but after 2 weeks there were more PMNs/unit area than in controls. The onset of measurable BrdU labeling was delayed in DFMO-treated animals, and the magnitude of labeling was significantly reduced. Increases in astrocyte and vessel numbers/mm2 were observed after a 2-week delay. At the site of implant, astrocytes from DFMO-treated dogs were significantly smaller than those from controls. Conclusions: There is substantial cell proliferation and infiltration in response to interstitial irradiation of normal brain, and these responses are significantly altered by DFMO treatment. Although the precise mechanisms by which DFMO exerts its effects in this model are not known, the results from this study suggest that modification of radiation

  4. Synthesis characterization and biological evaluation of a novel mixed ligand 99mTc complex as potential brain imaging agent

    International Nuclear Information System (INIS)

    One approach in the design of neutral oxotechnetium complexes is based on the simultaneous substitution of a tridentate dianionic ligand and a monodentate monoanionic coligand on a [Tc(V)O]+3 precursor. Following this ''mixed ligand'' concept, a novel 99mTc complex with N,N-bis(2-mercaptoethyl)-N'N'-diethylethylenediamine as ligand and 1-octanethiol as coligand is prepared and evaluated as potential brain radiopharmaceutical. Preparation of the complex at tracer level was accomplished by using 99mTc-glucoheptonate as precursor. The substitution was optimized and a coligand/ligand ratio of 5 was selected. Under this conditions the labeling yield was over 80% and a major product (with radiochemical purity > 80%) was isolated by HPLC methods and used for biological evaluation. Chemical characterization at carrier level was developed using the corresponding rhenium complex as structural model. The Re complex was also prepared by substitution method and isolated as a crystalline product. The crystals were characterized by UV-vis and IR spectra and elemental analysis. Results were consistent with the expected ReOLC structure. X ray crystallographic study demonstrated that the complex adopts a distorted trigonal bipyramidal geometry. The basal plane is defined by the SS atoms of the ligand and the oxo group, while the N of the ligand and the S of the colligand occupy the two apical positions. All sulphur atoms underwent ionization leading to the formation of a neutral compound. 99Tc complex was also prepared. Although it was not isolated due to the small amount of reagents employed, the HPLC profile was identical to the one observed for the rhenium complex suggesting the same chemical structure. Biodistribution in mice demonstrated early brain uptake, fast blood clearance, excretion through hepatobiliary system and a brain/blood ratio that increased significantly with time. (author)

  5. Synthesis and in vivo distribution in rat brain of 11C-labelled N-alkylated ADTN derivatives

    International Nuclear Information System (INIS)

    A method for the rapid production and purification of 11C-labelled N-alkylated derivatives of the dopamine agonist 2-amino-6,7-dihydroxytetralin (ADTN) is described. The label is introduced by N-methylation with no-carrier-added 11CH3I of the corresponding secondary amines via their lithium salts. Following systemic injection in rats a uniform distribution of radioactivity in the brain was found for both the labelled 2-(N-methyl-N-n-propylamino)- and 2-(N,N-dimethylamino)-6,7-dihydroxytetralin. (author)

  6. Loss of LRPPRC causes ATP synthase deficiency.

    Science.gov (United States)

    Mourier, Arnaud; Ruzzenente, Benedetta; Brandt, Tobias; Kühlbrandt, Werner; Larsson, Nils-Göran

    2014-05-15

    Defects of the oxidative phosphorylation system, in particular of cytochrome-c oxidase (COX, respiratory chain complex IV), are common causes of Leigh syndrome (LS), which is a rare neurodegenerative disorder with severe progressive neurological symptoms that usually present during infancy or early childhood. The COX-deficient form of LS is commonly caused by mutations in genes encoding COX assembly factors, e.g. SURF1, SCO1, SCO2 or COX10. However, other mutations affecting genes that encode proteins not directly involved in COX assembly can also cause LS. The leucine-rich pentatricopeptide repeat containing protein (LRPPRC) regulates mRNA stability, polyadenylation and coordinates mitochondrial translation. In humans, mutations in Lrpprc cause the French Canadian type of LS. Despite the finding that LRPPRC deficiency affects the stability of most mitochondrial mRNAs, its pathophysiological effect has mainly been attributed to COX deficiency. Surprisingly, we show here that the impaired mitochondrial respiration and reduced ATP production observed in Lrpprc conditional knockout mouse hearts is caused by an ATP synthase deficiency. Furthermore, the appearance of inactive subassembled ATP synthase complexes causes hyperpolarization and increases mitochondrial reactive oxygen species production. Our findings shed important new light on the bioenergetic consequences of the loss of LRPPRC in cardiac mitochondria. PMID:24399447

  7. The distribution of ATP within tomato (Lycopersicon esculentum Mill.) embryos correlates with germination whee as total ATP concentration does not

    NARCIS (Netherlands)

    Spoelstra, P.; Joosen, R.V.L.; Hilhorst, H.W.M.

    2002-01-01

    The distribution of ATP in tomato seeds was visualized by monitoring the luminescence of frozen sections on top of a gel containing all the components of the luciferase reaction, but excluding ATP. ATP was imaged in germinating tomato seeds at intervals of 3, 6, 17, 24 and 48 h and in seeds with pri

  8. Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

    OpenAIRE

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

    2009-01-01

    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 α and β subunits. Crystal structures of both α and β subunits that bind to the sub...

  9. Cellular proliferation and infiltration following interstitial irradiation of normal dog brain is altered by an inhibitor of polyamine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Fike, J.R.; Gobbel, G.T.; Chou, D. [Univ. of California, San Francisco, CA (United States)] [and others

    1995-07-15

    The objectives of this study were to quantitatively define proliferative and infiltrative cell responses after focal {sup 125}I irradiation of normal brain, and to determine the effects of an intravenous infusion of {alpha}-defluoromethylornithine (DFMO) on those responses. Adult beagle dogs were irradiated using high activity {sup 125}I sources. Cellular responses were quantified using a histomorphometric analysis. After radiation alone, cellular events included a substantial acute inflammatory response followed by increased BrdU labeling and progressive increases in numbers of capillaries and astrocytes. {alpha}-Difluoromethylornithine treatment significantly affected the measured cell responses. As in controls, an early inflammatory response was measured, but after 2 weeks there were more PMNs/unit area than in controls. The onset of measurable BrdU labeling was delayed in DFMO-treated animals, and the magnitude of labeling was significantly reduced. Increases in astrocyte and vessel numbers/mm{sup 2} were observed after a 2-week delay. At the site of implant, astrocytes from DFMO-treated dogs were significantly smaller than those from controls. There is substantial cell proliferation and infiltration in response to interstitial irradiation of normal brain, and these responses are significantly altered by DFMO treatment. Although the precise mechanisms by which DFMO exerts its effects in this model are not known, the results from this study suggest that modification of radiation injury may be possible by manipulating the response of normal cells to injury. 57 refs., 6 figs.

  10. Synthesis of [11C-methyl]-(-)-OSU6162, its regional brain distribution and some pharmacological effects of (-)-OSU6162 on the dopaminergic system studied in the rhesus monkey by positron emission tomography

    International Nuclear Information System (INIS)

    The labelling of the presynaptic dopamine receptor antagonist (-)-OSU6162, ((S)-(-)-3-(3-(methylsulfonyl)phenyl)-1-propylpiperidine) was performed by an alkylation with [11C]methyl iodide of the thio anion (-)-OSU1281, followed by a selective oxidation to the corresponding methyl sulfone, [11C-methyl]-(-)-OSU6162. The total radiochemical yield calculated from the produced [11C]carbon dioxide to final product was about 25% and the time of synthesis was in the range of 40 min from end of bombardment. The synthesis of the precursor, (-)-OSU1281, was performed from (-)-3PPP in a three-step synthesis. The regional brain distribution of (-)-OSU6162 radiolabelled with 11C was studied in rhesus monkeys by means of positron emission tomography, PET. [11C-Methyl]-(-)-OSU6162 was rapidly and uniformly distributed to gray matters of the brain, and no decrease of radioactivity uptake in the brain was seen after pretreatment with 1 to 3 mg/kg/h of (-)-OSU6162. The effect of doses of 1 to 3 mg/kg/h of (-)-OSU6162 on the dopamine binding was studied by PET using [11C-methyl]raclopride. Radioactivity in the striatum was significantly and dose-dependently decreased by (-)-OSU6162 (r = 0.88), supporting competition with dopamine for selective binding to dopamine receptors

  11. New novel mutation of the ATP7B gene in a family with Wilson disease.

    Science.gov (United States)

    Lee, Jun-Young; Kim, Young-Hyun; Kim, Tae-Woo; Oh, Sun-Young; Kim, Dal-Sik; Shin, Byoung-Soo

    2012-02-15

    Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The WD gene codes for a copper transporting P-type ATPase (ATP7B) are located on chromosome 13q14.3. Mutation of this gene disrupts copper homeostasis, resulting in the accumulation of copper in the liver, brain, kidneys and corneas and copper toxication at these sites. Since the detection of the WD gene in 1993, approximately 300 disease-specific muations have been identified. We recently evaluated a Korean family with WD. The proband, a 17-year-old boy, visited our hospital due to abnormal behaviors including generalized slow movement, dysphagia, drooling and ataxia. Laboratory results revealed decreases in serum copper and ceruloplasmin and an increase in urinary excretion of copper. He had liver cirrhosis, brain lesions and Kayser-Fleischer corenal rings. Molecular genetic analysis of the ATP7B gene demonstrated that he was heterozygous for deletion mutation c.2697_2723del27 in exon 11. Further study of family members revealed that his father and younger brother had the same mutation. The c.2697_2723del27 deletion mutation in exon 11 has not yet been reported as a causative muation of WD and is an in-frame deletion not expected to lead to a frame shift. Therefore, we report a novel mutation of the ATP7B gene in a family with WD. PMID:22075048

  12. ATP synthase of yeast mitochondria. Isolation of subunit j and disruption of the ATP18 gene.

    Science.gov (United States)

    Arnold, I; Pfeiffer, K; Neupert, W; Stuart, R A; Schägger, H

    1999-01-01

    The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase. PMID:9867807

  13. Anti-ATP synthase autoantibodies from patients with Alzheimer's disease reduce extracellular HDL level.

    Science.gov (United States)

    Vacirca, Davide; Barbati, Cristiana; Scazzocchio, Beatrice; Masella, Roberta; Rosano, Giuseppe; Malorni, Walter; Ortona, Elena

    2011-01-01

    Aside from being an integral protein involved in the synthesis and hydrolysis of ATP, Ecto-F1-ATPase plays a role in cholesterol homeostasis. We demonstrated the presence of autoantibodies to ecto-F1-ATPase (ASabs) in sera and cerebrospinal fluids from patients with Alzheimer's disease (AD). Herein we show that ASabs, unlike irrelevant antibodies, can increase cellular uptake of HDL, a risk factor for the development of AD, via a mechanism involving the prototypical function of ecto-F1-ATPase: the generation of ADP due to the hydrolysis of ATP. Piceatannol, a specific inhibitor ecto-F1-ATPase, completely hindered these effects. We hypothesize that ASabs could exert a pathogenetic role in AD. PMID:21677380

  14. Photosystem 0, a proposed ancestral photosystem without reducing power that synthesizes ATP during light-dark cycling

    CERN Document Server

    Muller, A W J

    2005-01-01

    Photosystem 0 is a proposed membrane-based mechanism for free energy gain as ATP from fluctuating light. The PS0 reaction centers have no reducing power: charge transport is only temporary. Light induces within the reaction centers metastable dipoles that generate a membrane potential that drives ATP synthesis while protons move through the ATPsynthase enzyme. After the decay of the dipole potential in the dark, the protons either (1) return across the membrane by conduction or (2) are pumped back by ATPsynthase, backwards active as ATPase at a higher H+/ATP ratio. PS0 constitutes a link to previously proposed free energy sources for early evolution that work on thermal cycling. Several contemporary photosynthetic phenomena may be relics of PS0.

  15. New fluorinated ligands for the dopamine transporter. Synthesis and first biological evaluation in pig and rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Krebs, B.; Sihver, W.; Coenen, H.H. [Forschungszentrum Juelich (Germany). Inst. fuer Nuklearchemie

    2004-07-01

    A change in the density of the dopamine transporter (DAT) is a widely accepted indicator for the integrity of the presynaptic nigrostriatal dopaminergic system. In spite of the advantageous properties of fluorine-18 for PET imaging up to now there is no suitable radiofluorinated DAT ligand available although numerous investigations have been performed by different working groups. Presently the SPECT ligand [{sup 123}I]FP-CIT known as DaTSCAN is the only commercially available DAT radiotracer for routine clinical use. The present study describes the syntheses of new fluorinated tropanes as potential DAT ligands (Figure) and their first in vitro evaluation in pig and rat brain. Several known DAT ligands are also synthesized and evaluated as standards for comparison. (orig.)

  16. Synthesis and radiolabelling-facilitated by Cu (1) of Para-Iodoamphetamine (PIPA): A radiopharmaceutical for brain perfusion imaging

    International Nuclear Information System (INIS)

    The ultimate purpose of this paper is to develop a method for routine production of Para-Iodoamphetamine (PIPA) from phenamine, which is produced formerly through Leukart-Wallach reaction and elemental iodine by an electrophilic iodination reaction. Verification of the final product as indeed P-IPA is performed using melting point, IR spectroscopy, GC chromatography, and NMR spectroscopy. Production of high quality P-IPA labeled with radioactive 131I by Isotopic Exchange Reaction facilitated by Cu (1) generated ,In situ, is described in this paper. This type of radiopharmaceutical is produced to develop a tracer which could be taken up by the brain. [131I]-PIPA is obtained with radiochemical yield greater than 85% radiochemical purity of final product [131I]-PIPA is assessed by use of paper chromatography with high resolution. (author)

  17. Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Hong-shuo SUN; Zhong-ping FENG

    2013-01-01

    ATP-sensitive potassium (KATP) channels are weak,inward rectifiers that couple metabolic status to cell membrane electrical activity,thus modulating many cellular functions.An increase in the ADP/ATP ratio opens KATP channels,leading to membrane hyperpolarization.KATP channels are ubiquitously expressed in neurons located in different regions of the brain,including the hippocampus and cortex.Brief hypoxia triggers membrane hyperpolarization in these central neurons.In vivo animal studies confirmed that knocking out the Kir6.2 subunit of the KATP channels increases ischemic infarction,and overexpression of the Kir6.2 subunit reduces neuronal injury from ischemic insults.These findings provide the basis for a practical strategy whereby activation of endogenous KATP channels reduces cellular damage resulting from cerebral ischemic stroke.KATP channel modulators may prove to be clinically useful as part of a combination therapy for stroke management in the future.

  18. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Jason Alan Gruenhagen

    2003-12-12

    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 Cd

  19. Brain serotonin synthesis in adult males characterized by physical aggression during childhood: a 21-year longitudinal study.

    Directory of Open Access Journals (Sweden)

    Linda Booij

    Full Text Available BACKGROUND: Adults exhibiting severe impulsive and aggressive behaviors have multiple indices of low serotonin (5-HT neurotransmission. It remains unclear though whether low 5-HT mediates the behavior or instead reflects a pre-existing vulnerability trait. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, positron emission tomography with the tracer alpha-[(11C]methyl-L-tryptophan ((11C-AMT was used to compare 5-HT synthesis capacity in two groups of adult males from a 21-year longitudinal study (mean age +/- SD: 27.1+/-0.7: individuals with a history of childhood-limited high physical aggression (C-LHPA; N = 8 and individuals with normal (low patterns of physical aggression (LPA; N = 18. The C-LHPA males had significantly lower trapping of (11C-AMT bilaterally in the orbitofrontal cortex and self-reported more impulsiveness. Despite this, in adulthood there were no group differences in plasma tryptophan levels, genotyping, aggression, emotional intelligence, working memory, computerized measures of impulsivity, psychosocial functioning/adjustment, and personal and family history of mood and substance abuse disorders. CONCLUSIONS/SIGNIFICANCE: These results force a re-examination of the low 5-HT hypothesis as central in the biology of violence. They suggest that low 5-HT does not mediate current behavior and should be considered a vulnerability factor for impulsive-aggressive behavior that may or may not be expressed depending on other biological factors, experience, and environmental support during development.

  20. Effect of the ATPase inhibitor protein IF1 on H+ translocation in the mitochondrial ATP synthase complex

    International Nuclear Information System (INIS)

    The H+ FoF1-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F1 moiety of the complex protrudes at the inner side of the membrane, the Fo sector spans the membrane reaching the outer side. The IF1 component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the FoF1-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF1 (residue 42-58) to the F1-α/β subunits. We have analyzed the effect of native purified IF1 the IF1-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K+] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted FoF1 complex. The results show that IF1, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F1 to the Fo side and in the opposite direction. Cross-linking of IF1 to F1-α/β subunits inhibits the ATP-driven H+ translocation but enhances H+ conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the FoF1 complex.

  1. Prenatal Exposure of Cypermethrin Induces Similar Alterations in Xenobiotic-Metabolizing Cytochrome P450s and Rate-Limiting Enzymes of Neurotransmitter Synthesis in Brain Regions of Rat Offsprings During Postnatal Development.

    Science.gov (United States)

    Singh, Anshuman; Mudawal, Anubha; Maurya, Pratibha; Jain, Rajeev; Nair, Saumya; Shukla, Rajendra K; Yadav, Sanjay; Singh, Dhirendra; Khanna, Vinay Kumar; Chaturvedi, Rajnish Kumar; Mudiam, Mohana K R; Sethumadhavan, Rao; Siddiqi, Mohammad Imran; Parmar, Devendra

    2016-08-01

    Oral administration of low doses of cypermethrin to pregnant Wistar rats led to a dose-dependent differences in the induction of xenobiotic-metabolizing cytochrome P450s (CYPs) messenger RNA (mRNA) and protein in brain regions isolated from the offsprings postnatally at 3 weeks that persisted up to adulthood. Similar alterations were observed in the expression of rate-limiting enzymes of neurotransmitter synthesis in brain regions of rat offsprings. These persistent changes were associated with alterations in circulating levels of growth hormone (GH), cognitive functions, and accumulation of cypermethrin and its metabolites in brain regions of exposed offsprings. Though molecular docking studies failed to identify similarities between the docked conformations of cypermethrin with CYPs and neurotransmitter receptors, in silico analysis identified regulatory sequences of CYPs in the promoter region of rate-limiting enzymes of neurotransmitter synthesis. Further, rechallenge of the prenatally exposed offsprings at adulthood with cypermethrin (p.o. 10 mg/kg × 6 days) led to a greater magnitude of alterations in the expression of CYPs and rate-limiting enzymes of neurotransmitter synthesis in different brain regions. These alterations were associated with a greater magnitude of decrease in the circulating levels of GH and cognitive functions in rechallenged offsprings. Our data has led us to suggest that due to the immaturity of CYPs in fetus or during early development, even the low-level exposure of cypermethrin may be sufficient to interact with the CYPs, which in turn affect the neurotransmission processes and may help in explaining the developmental neurotoxicity of cypermethrin. PMID:26115703

  2. Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

    Science.gov (United States)

    Feofilova, Maria; Howard, Jonathon

    Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

  3. Mitochondrial membrane potential and ATP production in primary disorders of ATP synthase

    Czech Academy of Sciences Publication Activity Database

    Vojtíšková, Alena; Ješina, Pavel; Kalous, Martin; Kaplanová, Vilma; Houštěk, Josef; Tesařová, M.; Fornůsková, D.; Zeman, J.; Dubot, A.; Godinot, C.

    2004-01-01

    Roč. 14, č. 1-2 (2004), s. 7-11. ISSN 1537-6524 R&D Projects: GA MZd NE6533; GA MŠk LN00A079 Institutional research plan: CEZ:AV0Z5011922 Keywords : ATP6 * membrane potential * mitochondrial diseases Subject RIV: ED - Physiology Impact factor: 0.464, year: 2004

  4. Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells

    International Nuclear Information System (INIS)

    Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical 'switchpoint' indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells

  5. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    International Nuclear Information System (INIS)

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation

  6. Effects of ATP on calcium binding to synaptic plasma membrane

    International Nuclear Information System (INIS)

    The release of labeled norepinephrine from preloaded synaptosomes requires the presence of potassium and calcium. ATP-dependent binding of calcium to synaptic plasma membranes (SPM) may provide a means of maintaining the cation in a readily available pool for the triggering of transmitter release. A high Ca-binding capacity was demonstrated in SPM. The Km for calcium is 5.5 X 10(-5) M. The dependence of the system on the gamma phosphate of ATP was demonstrated by an increase in Ca-binding with increasing ATP concentration and by competitive inhibition of binding by ADP and AMP. Magnesium is also required for ATP-dependent Ca-binding. The optimum pH for the Ca binding was 7.0. Pretreatment of SPM with phospholipase A2 lowered the binding capacity. Sulfhydryl groups are also critical for ATP-dependent Ca binding to occur. A model for ATP-dependent Ca-binding was proposed

  7. Willed-movement training reduces brain damage and enhances synaptic plasticity related proteins synthesis after focal ischemia.

    Science.gov (United States)

    Nie, Jingjing; Yang, Xiaosu; Tang, Qingping; Shen, Qin; Li, Simin

    2016-01-01

    It has been wildly accepted that willed movement(WM) training promotes neurological rehabilitation in patients with stroke. However, it was not clear whether the effect of WM is better than other forms of exercise. The purpose of this study is to assess different effects of WM and other forms of exercise on rats with focal ischemia. The subjects are all had right middle cerebral artery occlusion (MCAO) surgery and randomly allocated to three groups of training and one control group with no training. Infarct volume by 2,3,5-triphenyltetrazolium chloride (TTC) dye, expression of PICK1 and synaptophysin in cerebral cortex and striatum of injured side by western blotting and immunofluorescence performed are analyzed. Exercise has done respectively on rats in each group for 15 days and 30 days. Compared with the control group, the brain damage is reduced in other groups after 15 days exercise. The protein expressions levels of synaptophysin and PICK1 are upregulated after exercise. Concentration of PICK1 protein in WM is greater than other exercise groups, and the expression of synaptophysin in WM and SM groups are higher than EM groups. The number of PICK1 positive cells, synaptophysin and PICK1 co-positive cells are increased by exercise. Synaptophysin is widely distributed in cortex surrounding the injury area in WM and EM. It is indicated in our result that willed-movement training is the most effective intervention in enhancing the PICK1-mediated synaptic plasticity in the area adjacent to the damage region of ischemic rats. PMID:26556240

  8. Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

    OpenAIRE

    Wachter, C.; Schatz, G.; Glick, B S

    1994-01-01

    ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix ...

  9. A new system for rapid measurement of ATP.

    Science.gov (United States)

    Shu, B; Zhou, Y; Ren, S

    1997-01-01

    The paper introduces a new type instrument for rapid measuring ATP. The system consists of a micromodule ATP sensor and an instrument for measuring weak light transmitted by optic fiber. The micromodule ATP sensor mainly is composed of enzyme membrane, a probe and a bundle of optic fiber. The instrument measuring weak light consists of photomultiplier, high voltage power, pulse amplifier and counter. The instrument was characterized by simple structure, small size, rapid response time (times, CV time (> 3 months). PMID:9812776

  10. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    Science.gov (United States)

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  11. ATP Freisetzung aus neutrophilen Granulozyten durch "Connexin 43 hemichannels"

    OpenAIRE

    Küper, Natalie

    2008-01-01

    Extracellular ATP liberated during hypoxia and inflammation can either signal directly on purinergic receptors or can activate adenosine receptors following phosphohydrolysis to adenosine. Given the association of polymorphonuclear leukocytes (PMNs) with adenine-nucleotide/nucleoside signaling in the inflammatory milieu, we hypothesized that PMNs are a source of extracellular ATP. Initial studies using high-performance liquid chromatography and luminometric ATP detection assays revealed that ...

  12. Carbon and energy metabolism of atp mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    The membrane-bound H+-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth...... rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion...

  13. Effect of irradiation on bioluminescence spectrum of microbial ATP

    International Nuclear Information System (INIS)

    The effect of irradiation on bioluminescence spectrum of dehydrated cabbage microbial ATP was studied. The results showed that the spectral bandwidth of ATP standard was from 490 to 640 nm and the peak wavelength was at 563 nm. The spectral bandwidths of irradiated dehydrated cabbage microbial ATP and CK did not change. Peak wavelengths of dehydrated cabbage irradiated at different dosages were not significantly different from that of CK. The peaks of bioluminescence spectrum of irradiated samples were higher than that of CK, which may be because of the increasing concentration of ATP, and this effect would be kept for quite a long time after irradiation. (authors)

  14. The biphasic response of rat vesical smooth muscle to ATP.

    OpenAIRE

    Bolego, C; Pinna, C.; Abbracchio, M. P.; Cattabeni, F.; Puglisi, L.

    1995-01-01

    1. Adenosine-5'-triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2x- or G-protein coupled P2Y-purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol-prestimulated tissues. 2. ATP exerted a clear concentration-dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contr...

  15. Brain Basics

    Medline Plus

    Full Text Available ... News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  16. Brain Basics

    Science.gov (United States)

    ... News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... Brain Basics provides information on how the brain works, how mental illnesses are disorders of the brain, ... learning more about how the brain grows and works in healthy people, and how normal brain development ...

  18. Design, synthesis, and evaluation of cisplatin-containing EGFR targeting bioconjugates as potential therapeutic agents for brain tumors

    Science.gov (United States)

    Barth, Rolf F; Wu, Gong; Meisen, W Hans; Nakkula, Robin J; Yang, Weilian; Huo, Tianyao; Kellough, David A; Kaumaya, Pravin; Turro, Claudia; Agius, Lawrence M; Kaur, Balveen

    2016-01-01

    The aim of this study was to evaluate four different platinated bioconjugates containing a cisplatin (cis-diamminedichloroplatinum [cis-DDP]) fragment and epidermal growth factor receptor (EGFR)-targeting moieties as potential therapeutic agents for the treatment of brain tumors using a human EGFR-expressing transfectant of the F98 rat glioma (F98EGFR) to assess their efficacy. The first two bioconjugates employed the monoclonal antibody cetuximab (C225 or Erbitux®) as the targeting moiety, and the second two used genetically engineered EGF peptides. C225-G5-Pt was produced by reacting cis-DDP with a fifth-generation polyamidoamine dendrimer (G5) and then linking it to C225 by means of two heterobifunctional reagents. The second bioconjugate (C225-PG-Pt) employed the same methodology except that polyglutamic acid was used as the carrier. The third and fourth bioconjugates used two different EGF peptides, PEP382 and PEP455, with direct coordination to the Pt center of the cis-DDP fragment. In vivo studies with C225-G5-Pt failed to demonstrate therapeutic activity following intracerebral (ic) convection-enhanced delivery (CED) to F98EGFR glioma-bearing rats. The second bioconjugate, C225-PG-Pt, failed to show in vitro cytotoxicity. Furthermore, because of its high molecular weight, we decided that lower molecular weight peptides might provide better targeting and microdistribution within the tumor. Both PEP382-Pt and PEP455-Pt bioconjugates were cytotoxic in vitro and, based on this, a pilot study was initiated using PEP455-Pt. The end point for this study was tumor size at 6 weeks following tumor cell implantation and 4 weeks following ic CED of PEP455-Pt to F98 glioma-bearing rats. Neuropathologic examination revealed that five of seven rats were either tumor-free or only had microscopic tumors at 42 days following tumor implantation compared to a mean survival time of 20.5 and 26.3 days for untreated controls. In conclusion, we have succeeded in reformatting the

  19. ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

    OpenAIRE

    Zeng, Xiaomei; Barros, Mario H.; Shulman, Theodore; Tzagoloff, Alexander

    2008-01-01

    We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of...

  20. Rat Mitochondrion-Neuron Focused Microarray (rMNChip and Bioinformatics Tools for Rapid Identification of Differential Pathways in Brain Tissues

    Directory of Open Access Journals (Sweden)

    Yan A. Su, Qiuyang Zhang, David M. Su, Michael X. Tang

    2011-01-01

    Full Text Available Mitochondrial function is of particular importance in brain because of its high demand for energy (ATP and efficient removal of reactive oxygen species (ROS. We developed rat mitochondrion-neuron focused microarray (rMNChip and integrated bioinformatics tools for rapid identification of differential pathways in brain tissues. rMNChip contains 1,500 genes involved in mitochondrial functions, stress response, circadian rhythms and signal transduction. The bioinformatics tool includes an algorithm for computing of differentially expressed genes, and a database for straightforward and intuitive interpretation for microarray results. Our application of these tools to RNA samples derived from rat frontal cortex (FC, hippocampus (HC and hypothalamus (HT led to the identification of differentially-expressed signal-transduction-bioenergenesis and neurotransmitter-synthesis pathways with a dominant number of genes (FC/HC = 55/6; FC/HT = 55/4 having significantly (p<0.05, FDR<10.70% higher (≥1.25 fold RNA levels in the frontal cortex than the others, strongly suggesting active generation of ATP and neurotransmitters and efficient removal of ROS. Thus, these tools for rapid and efficient identification of differential pathways in brain regions will greatly facilitate our systems-biological study and understanding of molecular mechanisms underlying complex and multifactorial neurodegenerative diseases.

  1. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    Directory of Open Access Journals (Sweden)

    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  2. Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.

    Science.gov (United States)

    Fishburn, James; Galburt, Eric; Hahn, Steven

    2016-06-17

    Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction. PMID:27129284

  3. Prohibitins Interact Genetically with Atp23, a Novel Processing Peptidase and Chaperone for the F1FO-ATP Synthase

    OpenAIRE

    Osman, Christof; Wilmes, Claudia; Tatsuta, Takashi; Langer, Thomas

    2007-01-01

    The generation of cellular energy depends on the coordinated assembly of nuclear and mitochondrial-encoded proteins into multisubunit respiratory chain complexes in the inner membrane of mitochondria. Here, we describe the identification of a conserved metallopeptidase present in the intermembrane space, termed Atp23, which exerts dual activities during the biogenesis of the F1FO-ATP synthase. On one hand, Atp23 serves as a processing peptidase and mediates the maturation of the mitochondrial...

  4. Design, synthesis, and evaluation of cisplatin-containing EGFR targeting bioconjugates as potential therapeutic agents for brain tumors

    Directory of Open Access Journals (Sweden)

    Barth RF

    2016-05-01

    Full Text Available Rolf F Barth,1 Gong Wu,1 W Hans Meisen,2 Robin J Nakkula,1 Weilian Yang,1 Tianyao Huo,1 David A Kellough,1 Pravin Kaumaya,3–5 Claudia Turro,6 Lawrence M Agius,7 Balveen Kaur2 1Department of Pathology, 2Department of Neurological Surgery, 3Department of Obstetrics and Gynecology, 4Department of Molecular and Cellular Biochemistry, 5Department of Microbiology, 6Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA; 7Department of Pathology, Mater Dei Hospital, University of Malta Medical School, Msida, Malta Abstract: The aim of this study was to evaluate four different platinated bioconjugates containing a cisplatin (cis-diamminedichloroplatinum [cis-DDP] fragment and epidermal growth factor receptor (EGFR-targeting moieties as potential therapeutic agents for the treatment of brain tumors using a human EGFR-expressing transfectant of the F98 rat glioma (F98EGFR to assess their efficacy. The first two bioconjugates employed the monoclonal antibody cetuximab (C225 or Erbitux® as the targeting moiety, and the second two used genetically engineered EGF peptides. C225-G5-Pt was produced by reacting cis-DDP with a fifth-generation polyamidoamine dendrimer (G5 and then linking it to C225 by means of two heterobifunctional reagents. The second bioconjugate (C225-PG-Pt employed the same methodology except that polyglutamic acid was used as the carrier. The third and fourth bioconjugates used two different EGF peptides, PEP382 and PEP455, with direct coordination to the Pt center of the cis-DDP fragment. In vivo studies with C225-G5-Pt failed to demonstrate therapeutic activity following intracerebral (ic convection-enhanced delivery (CED to F98EGFR glioma-bearing rats. The second bioconjugate, C225-PG-Pt, failed to show in vitro cytotoxicity. Furthermore, because of its high molecular weight, we decided that lower molecular weight peptides might provide better targeting and microdistribution within the tumor. Both PEP

  5. Structural models of the human copper P-type ATPases ATP7A and ATP7B

    DEFF Research Database (Denmark)

    Gourdon, Pontus Emanuel; Sitsel, Oleg; Karlsen, Jesper Lykkegaard;

    2012-01-01

    The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B...... Legionella pneumophila. The models and sequence analyses show that the domains and residues involved in the catalytic phosphorylation events and copper transfer are highly conserved. In addition, there are only minor differences in the core structures of the two human proteins and the bacterial template...

  6. Modelling the ATP production in mitochondria

    CERN Document Server

    Saa, Alberto

    2012-01-01

    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 correct some inaccuracies in the BPLS original approximations 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. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium ${\\rm Ca}_{\\rm c}$ and the substrate fructose 1,6-bisphosphate FBP. The same effect of calcium saturation reported for the original BPLS model is observed here. We find out, however, an interesting non-stationary effect: the inertia of the model tends to increase considerably for high concentrations of ...

  7. Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

    Science.gov (United States)

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems. PMID:27295623

  8. Synthesis of new radiotracers

    International Nuclear Information System (INIS)

    The brain's sensibility besides to the rigorous selectivity of changes taking place on brain's barriers leads us to synthesis specifics radiotracers based on diamine ethylene and marked with technetium radioisotope to form a radiotracer able to pass these barriers and diagnose illnesses in an early stage. These radiotracers are tested by a biodistribution on a small animal to be ratified. (Author)

  9. A new type of Na(+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

    Directory of Open Access Journals (Sweden)

    Sarah Schulz

    Full Text Available The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F₀-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

  10. An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

    International Nuclear Information System (INIS)

    Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn2+ inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn2+ inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn2+ inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F1F0 ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn2+ inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

  11. Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1β Release.

    Science.gov (United States)

    Hecker, Andreas; Küllmar, Mira; Wilker, Sigrid; Richter, Katrin; Zakrzewicz, Anna; Atanasova, Srebrena; Mathes, Verena; Timm, Thomas; Lerner, Sabrina; Klein, Jochen; Kaufmann, Andreas; Bauer, Stefan; Padberg, Winfried; Kummer, Wolfgang; Janciauskiene, Sabina; Fronius, Martin; Schweda, Elke K H; Lochnit, Günter; Grau, Veronika

    2015-09-01

    IL-1β is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1β plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1β release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1β synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1β by caspase-1, and release of mature IL-1β. Mechanisms controlling IL-1β release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1β release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1β and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host. PMID:26202987

  12. Mutation analysis of ATP13A2 in early-onset parkinsonism patients

    Institute of Scientific and Technical Information of China (English)

    Yuping Ning; Hiroyuki Tomiyama; Yuanzhe Li; Manabu Funayama; Hiroyo Yoshino; Shigeto Sato; Yoshikuni Mizuno; Nobutaka Hattori

    2008-01-01

    BACKGROUND: A recent study has found that ATP13A2 is the causative gene for PARK9-linked autosomal recessive early-onset parkinsonism, described previously in Jordanian and Chilean families (Kufor-Rakeb syndrome). OBJECTIVE: To screen eastern Asian patients with early-onset parkinsonism for mutations in ATP13A2 and to describe positron emission tomography (PET) findings of PARK9-linked parkinsonism.DESIGN, TIME AND SETTING: In total, 117 patients were selected from the Department of Neurology, Juntendo University, from February 2003 to October 2006, for this molecular genetics and case-control study.PARTICIPANTS: The patients with parkinsonism consist of two cohorts. Ninety four patients with onset age of less than 30 years were selected for the first cohort. They included 49 males and 44 females, comprising 73 Japanese, 9 Korean, 8 Taiwanese, and 4 Mainland Chinese. Eleven patients had parkinsonism complicated with dementia, 15 patients had family histories of parkinsonism (including 2 families), and 5 patients were from consanguineous parents (including one family). The second cohort of 23 patients was composed of patients with consanguineous parents (n = 15) or who had affected siblings (n = 6) or both (n = 2), but the age at onset ranged from 30 to 50 years.METHODS: In 117 patients with parkinsonism, direct sequencing of ATP13A2 exons 13, 16, and 26, in which mutations had been reported previously, were performed. Sequencing was also performed in all 29 exons, including splice sites, in 28 probands who showed homozygosity at the PARK9 locus by haplotype analysis. Mutation analysis was also performed in 150 normal people. Linkage analysis was performed on all 3 parkinsonism families using short tandem repeat markers flanking the PARK9 locus. For patients who had ATP13A2 mutation, we performed brain MRI and 18F-dopa PET scans.MAIN OUTCOME MEASURES: ATP13A2 DNA sequence, 18F-dopa PET scan and brain MRI findings.RESULTS: A novel F182L mutation in a consanguineous

  13. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, 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-1β 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 proinflammatory 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.

  14. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko;

    2011-01-01

    BACKGROUND: Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene. ME...

  15. ATP measurements for monitoring microbial drinking water quality

    DEFF Research Database (Denmark)

    Vang, Óluva Karin

    carrying molecule in living cells, thus ATP can be used as a parameter for microbial activity. ATP is extracted from cells through cell lysis and subsequently assayed with the luciferase enzyme and its substrate luciferin, resulting in bioluminescence, i.e. light emission which can be quantified...

  16. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Sørensen, Mette Aaskov; Strøbech, Lotte Bjørg;

    2008-01-01

    Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle...... cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated...... pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration...

  17. ATP storage and uptake by isolated pancreatic zymogen granules

    DEFF Research Database (Denmark)

    Haanes, Kristian Agmund; Novak, Ivana

    2010-01-01

    ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism for...... ATP transport into the ZG. ZG were isolated and the ATP content was measured using luciferin/luciferase assays and was related to protein in the sample. The estimate of ATP concentration in freshly isolated granules was 40-120 µM. The ATP uptake had an apparent Km value of 4.9±2.1 mM when granules...... were incubated without Mg2+ and a Km value of 0.47±0.05 mM in the presence of Mg2+, both in pH 6.0 buffers. The uptake of ATP was significantly higher at pH 7.2 compared with pH 6.0 solutions. The anion transport blockers DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate) and Evans Blue inhibited ATP...

  18. K(ATP) channel openers in the trigeminovascular system

    DEFF Research Database (Denmark)

    Ploug, K B; Amrutkar, D V; Baun, M;

    2012-01-01

    The ATP-sensitive K(+) (K(ATP)) channel openers levcromakalim and pinacidil are vasodilators that induce headache in healthy people. The neuropeptide calcitonin gene-related peptide (CGRP) induces headache in healthy people and migraine in migraineurs, potentially through a mechanism that involve...

  19. An ATP transport system in the intracellular bacterium, Bdellovibrio bacteriovorus 109J.

    OpenAIRE

    Ruby, E G; McCabe, J B

    1986-01-01

    The intracellularly growing bacterium Bdellovibrio bacteriovorus 109J transports intact ATP by a specific, energy-requiring process. ATP transport does not involve either an ADP-ATP or an AMP-ATP exchange mechanism but, instead, has characteristics of an active transport permease. Kinetically distinct systems for ATP transport are expressed by the two developmental stages of the bdellovibrio life cycle.

  20. Mitochondrial free radical overproduction due to respiratory chain impairment in the brain of a mouse model of Rett syndrome: protective effect of CNF1.

    Science.gov (United States)

    De Filippis, Bianca; Valenti, Daniela; de Bari, Lidia; De Rasmo, Domenico; Musto, Mattia; Fabbri, Alessia; Ricceri, Laura; Fiorentini, Carla; Laviola, Giovanni; Vacca, Rosa Anna

    2015-06-01

    Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and

  1. Phylogenetic analysis of the thylakoid ATP/ADP carrier reveals new insights into its function restricted to green plants

    Directory of Open Access Journals (Sweden)

    Cornelia eSpetea

    2012-01-01

    Full Text Available ATP is the common energy currency of cellular metabolism in all living organisms. Most of them synthesize ATP in the cytosol or on the mitochondrial inner membrane, whereas land plants, algae and cyanobacteria also produce it on the thylakoid membrane during the light-dependent reactions of photosynthesis. From the site of synthesis, ATP is transported to the site of utilization via intracellular membranes transporters. One major type of ATP transporter is represented by the mitochondrial ADP/ATP carrier family. Here we review a recently characterized member, namely the thylakoid ATP/ADP carrier from Arabidopsis thaliana (AtTAAC. Thus far, no orthologues of this carrier have been characterized in other organisms, although similar sequences can be recognized in many sequenced genomes. Protein Sequence database searches and phylogenetic analyses indicate the absence of TAAC in cyanobacteria and its appearance early in the evolution of photosynthetic eukaryotes. The TAAC clade is composed of carriers found in land plants and some green algae, but no proteins from other photosynthetic taxa, such as red algae, brown algae and diatoms. This implies that TAAC-like sequences arose only once before the divergence of green algae and land plants. Based on these findings, it is proposed that TAAC may have evolved in response to the need of a new activity in higher photosynthetic eukaryotes. This activity may provide the energy to drive reactions during biogenesis and turnover of photosynthetic complexes, which are heterogenously distributed in a thylakoid membrane system composed of appressed and non-appressed regions.

  2. Fatty acid synthesis by spinach chloroplasts, 3

    International Nuclear Information System (INIS)

    The modes of actions of photosynthetic inhibitors on photosynthesis and fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acid synthesis and photophosphorylation to the same extent, suggesting dependence of fatty acid synthesis on photosynthesis. The same was also the case with FCCP, a photophosphorylation uncoupler. In contrast, NH4Cl and phlorizin at concentrations completely suppressing ATP formation, only partially inhibited the fatty acid synthesis. These facts suggest that a certain level of high-energy intermediate (state) is responsible for the light enhancement of fatty acid synthesis. This idea is further supported by the fact that the partial inhibition of fatty acid synthesis by NH4Cl was relieved by addition of DCCD at low concentrations suppressing the ATP formation but not completely destroying the high energy intermediate. The lag period in the initial period of fatty acid synthesis was shortened by preillumination of chloroplasts, even in the absence of ADP. This indicates that the light dependent fatty acid synthesis is closely associated with the high-energy intermediate (state), but not directly with ATP formation by photophosphorylation. (author)

  3. Amperometric ATP biosensor based on polymer entrapped enzymes.

    Science.gov (United States)

    Kueng, Angelika; Kranz, Christine; Mizaikoff, Boris

    2004-05-15

    A dual enzyme electrode for the detection of adenosine-5'-triphosphate (ATP) at physiologically relevant pH levels was developed by co-immobilization of the enzymes glucose oxidase (GOD) and hexokinase (HEX) using pH-shift induced deposition of enzyme containing polymer films. Application of a simple electrochemical procedure for the co-immobilization of the enzymes at electrode surfaces exhibits a major improvement of sensitivity, response time, reproducibility, and ease of fabrication of ATP biosensors. Competition between glucose oxidase and hexokinase for the substrate glucose involving ATP as a co-substrate allows the determination of ATP concentrations. Notable control on the immobilization process enables fabrication of micro biosensors with a diameter of 25 microm. The presented concept provides the technological basis for a new generation of fast responding, sensitive, and robust biosensors for the detection of ATP at physiological pH values with a detection limit of 10 nmol l(-1). PMID:15046763

  4. Brain herniation

    Science.gov (United States)

    ... herniation; Uncal herniation; Subfalcine herniation; Tonsillar herniation; Herniation - brain ... Brain herniation occurs when something inside the skull produces pressure that moves brain tissues. This is most ...

  5. Single-step synthesis of [18F]haloperidol from the chloro-precursor and its applications in PET imaging of a cat's brain

    International Nuclear Information System (INIS)

    We have established a convenient synthesis process for the synthesis of [18F]haloperidol using a single-step 18F - for -Cl exchange reaction and a new elution system for the preparative high performance liquid chromatography (HPLC) using C18 bonded vinylalcohol copolymer gel (ODP) and a basic eluent. We successfully applied the product to cat-PET study and got clear images of the striatum, showing the usefulness of this synthesis. (author)

  6. Expression of ATP7B in normal human liver

    Directory of Open Access Journals (Sweden)

    D Fanni

    2009-06-01

    Full Text Available ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

  7. EFFECTS OF HYPERTHERMIA AND HYPERTHERMIA PLUS MICROWAVES ON RAT BRAIN ENERGY METABOLISM

    Science.gov (United States)

    The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effects of temperature on adenosine triphosphate concentration (ATP) and creatine phosphate concentration (CP) was determi...

  8. Local release of ATP into the arterial inflow and venous drainage of human skeletal muscle: insight from ATP determination with the intravascular microdialysis technique

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Thaning, Pia; Nyberg, Michael Permin;

    2011-01-01

    Intraluminal ATP could play an important role in the local regulation of skeletal muscle blood flow, but the stimuli that cause ATP release and the levels of plasma ATP in vessels supplying and draining human skeletal muscle remain unclear. To gain insight into the mechanisms by which ATP is...... released into plasma, we measured plasma [ATP] with the intravascular microdialysis technique at rest and during dynamic exercise (normoxia and hypoxia), passive exercise, thigh compressions and arterial ATP, tyramine and ACh infusion in a total of 16 healthy young men. Femoral arterial and venous[ATP...

  9. Application of 31P MR spectroscopy to the brain tumors

    International Nuclear Information System (INIS)

    To evaluate the clinical feasibility and obtain useful parameters of 31P magnetic resonance spectroscopy (MRS) study for making the differential diagnosis of brain tumors. Twenty-eight patients with brain tumorous lesions (22 cases of brain tumor and 6 cases of abscess) and 11 normal volunteers were included. The patients were classified into the astrocytoma group, lymphoma group, metastasis group and the abscess group. We obtained the intracellular pH and the metabolite ratios of phosphomonoesters/phosophodiesters (PME/PDE), PME/inorganic phosphate (Pi), PDE/Pi, PME/adenosine triphosphate (ATP), PDE/ATP, PME/phosphocreatine (PCr), PDE/PCr, PCr/ATP, PCr/Pi, and ATP/Pi, and evaluated the statistical significances. The brain tumors had a tendency of alkalization (pH = 7.28 ± 0.27, p = 0.090), especially the pH of the lymphoma was significantly increased (pH = 7.45 ± 0.32, p = 0.013). The brain tumor group showed increased PME/PDE ratio compared with that in the normal control group (p 0.012). The ratios of PME/PDE, PDE/Pi, PME/PCr and PDE/PCr showed statistically significant differences between each brain lesion groups (p 1'P MRS, and the pH, PME/PDE, PDE/Pi, PME/PCr, and PDE/PCr ratios are helpful for differentiating among the different types of brain tumors.

  10. Brain Basics

    Medline Plus

    Full Text Available ... Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies show that brain growth in children with autism ...

  11. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  12. Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: Magnesium provides a bridge for ATP to fuel unwinding

    OpenAIRE

    Frick, David N.; Banik, Sukalyani; Rypma, Ryan S.

    2006-01-01

    This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus. Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the 4 rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These valu...

  13. Sulfonylurea Receptor 1 Contributes to the Astrocyte Swelling and Brain Edema in Acute Liver Failure

    OpenAIRE

    Jayakumar, A.R.; Valdes, V.; Tong, X. Y.; Shamaladevi, N.; W Gonzalez; Norenberg, M.D.

    2014-01-01

    Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, non-selective cation channel (NCCa-ATP ch...

  14. Authentic role of ATP signaling in micturition reflex.

    Science.gov (United States)

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-01

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2(-/-) and P2X3(-/-) mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex. PMID:26795755

  15. The origin of cytosolic ATP in photosynthetic cells.

    Science.gov (United States)

    Gardeström, Per; Igamberdiev, Abir U

    2016-07-01

    In photosynthetically active cells, both chloroplasts and mitochondria have the capacity to produce ATP via photophosphorylation and oxidative phosphorylation, respectively. Thus, theoretically, both organelles could provide ATP for the cytosol, but the extent, to which they actually do this, and how the process is regulated, both remain unclear. Most of the evidence discussed comes from experiments with rapid fractionation of isolated protoplasts subjected to different treatments in combination with application of specific inhibitors. The results obtained indicate that, under conditions where ATP demand for photosynthetic CO2 fixation is sufficiently high, the mitochondria supply the bulk of ATP for the cytosol. In contrast, under stress conditions where CO2 fixation is severely limited, ATP will build up in chloroplasts and it can then be exported to the cytosol, by metabolite shuttle mechanisms. Thus, depending on the conditions, either mitochondria or chloroplasts can supply the bulk of ATP for the cytosol. This supply of ATP is discussed in relation to the idea that mitochondrial functions may be tuned to provide an optimal environment for the chloroplast. By balancing cellular redox states, mitochondria can contribute to an optimal photosynthetic capacity. PMID:27087668

  16. ATP and potassium ions: a deadly combination for astrocytes

    Science.gov (United States)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

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

    OpenAIRE

    Jaramillo, Michael L; Abanto, Michel; Quispe, Ruth L; Calderón, Julio; del Valle, Luís J; Talledo, Miguel; Ramírez, Pablo

    2012-01-01

    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 ...

  18. Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis

    OpenAIRE

    1989-01-01

    The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K+ channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K+ channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (wit...

  19. Missense mutation in the ATPase, aminophospholipid transporter protein ATP8A2 is associated with cerebellar atrophy and quadrupedal locomotion

    Science.gov (United States)

    Emre Onat, Onur; Gulsuner, Suleyman; Bilguvar, Kaya; Nazli Basak, Ayse; Topaloglu, Haluk; Tan, Meliha; Tan, Uner; Gunel, Murat; Ozcelik, Tayfun

    2013-01-01

    Cerebellar ataxia, mental retardation and dysequilibrium syndrome is a rare and heterogeneous condition. We investigated a consanguineous family from Turkey with four affected individuals exhibiting the condition. Homozygosity mapping revealed that several shared homozygous regions, including chromosome 13q12. Targeted next-generation sequencing of an affected individual followed by segregation analysis, population screening and prediction approaches revealed a novel missense variant, p.I376M, in ATP8A2. The mutation lies in a highly conserved C-terminal transmembrane region of E1 E2 ATPase domain. The ATP8A2 gene is mainly expressed in brain and development, in particular cerebellum. Interestingly, an unrelated individual has been identified, in whom mental retardation and severe hypotonia is associated with a de novo t(10;13) balanced translocation resulting with the disruption of ATP8A2. These findings suggest that ATP8A2 is involved in the development of the cerebro-cerebellar structures required for posture and gait in humans. PMID:22892528

  20. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our...... dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting...

  1. Characterisation of ATP analogues to cross-link and label P2X receptors

    OpenAIRE

    Agboh, Kelvin C.; Andrew J. Powell; Evans, Richard J.

    2009-01-01

    P2X receptors are a distinct family of ATP-gated ion channels with a number of physiological roles ranging from smooth muscle contractility to the regulation of blood clotting. In this study we determined whether the UV light-reactive ATP analogues 2-azido ATP, ATP azidoanilide (ATP-AA) and 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) can be used to label the ATP binding site of P2X1 receptors. These analogues were agonists, and in patch clamp studies evoked inward currents from HEK293 cells stably...

  2. ATP, a partial agonist for the P2Z receptor of human lymphocytes

    OpenAIRE

    Gargett, Caroline E.; Cornish, Jean E; Wiley, James S.

    1997-01-01

    Although extracellular adenosine 5′-triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba2+ and ethidium+. We have quantified the influx of ethidium+ into lymphocytes produced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATPγS, studied competition between ATP and BzATP and investigated the effects of KN-62, a new and p...

  3. ATP-induced [Ca2+]i changes and depolarization in GH3 cells

    OpenAIRE

    Chung, Hae Sook; Park, Kyu Sang; Cha, Seung Kyu; Kong, In Deok; Lee, Joong Woo

    2000-01-01

    Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca2+ signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line.BzATP- and ATP-induced [Ca2+]i responses had EC50 values of 18 and 651 μM, respectively. The maximal response to ATP was only 59±8% of that for BzATP. The BzATP-induced [Ca2+...

  4. Synthesis and Preclinical Evaluation of Three Novel Fluorine-18 Labeled Radiopharmaceuticals for P-Glycoprotein PET Imaging at the Blood-Brain Barrier

    NARCIS (Netherlands)

    Savolainen, Heli; Cantore, Mariangela; Colabufo, Nicola A.; Elsinga, Philip H.; Windhorst, Albert D.; Luurtsema, Gert

    2015-01-01

    P-Glycoprotein (P-gp), along with other transporter proteins at the blood brain barrier (BBB), limits the entry of many pharmaceuticals into the brain. Altered P-gp function has been found in several neurological diseases. To study the P-gp function, many positron emission tomography (PET) radiophar

  5. GABA transmission via ATP-dependent K+ channels regulates α-synuclein secretion in mouse striatum.

    Science.gov (United States)

    Emmanouilidou, Evangelia; Minakaki, Georgia; Keramioti, Maria V; Xylaki, Mary; Balafas, Evangelos; Chrysanthou-Piterou, Margarita; Kloukina, Ismini; Vekrellis, Kostas

    2016-03-01

    α-Synuclein is readily released in human and mouse brain parenchyma, even though the normal function of the secreted protein has not been yet elucidated. Under pathological conditions, such as in Parkinson's disease, pathologically relevant species of α-synuclein have been shown to propagate between neurons in a prion-like manner, although the mechanism by which α-synuclein transfer induces degeneration remains to be identified. Due to this evidence extracellular α-synuclein is now considered a critical target to hinder disease progression in Parkinson's disease. Given the importance of extracellular α-synuclein levels, we have now investigated the molecular pathway of α-synuclein secretion in mouse brain. To this end, we have identified a novel synaptic network that regulates α-synuclein release in mouse striatum. In this brain area, the majority of α-synuclein is localized in corticostriatal glutamatergic terminals. Absence of α-synuclein from the lumen of brain-isolated synaptic vesicles suggested that they are unlikely to mediate its release. To dissect the mechanism of α-synuclein release, we have used reverse microdialysis to locally administer reagents that locally target specific cellular pathways. Using this approach, we show that α-synuclein secretion in vivo is a calcium-regulated process that depends on the activation of sulfonylurea receptor 1-sensitive ATP-regulated potassium channels. Sulfonylurea receptor 1 is distributed in the cytoplasm of GABAergic neurons from where the ATP-dependent channel regulates GABA release. Using a combination of specific agonists and antagonists, we were able to show that, in the striatum, modulation of GABA release through the sulfonylurea receptor 1-regulated ATP-dependent potassium channels located on GABAergic neurons controls α-synuclein release from the glutamatergic terminals through activation of the presynaptic GABAB receptors. Considering that sulfonylurea receptors can be selectively targeted, our

  6. Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion.

    Science.gov (United States)

    Kirby, Brett S; Hanna, Gabi; Hendargo, Hansford C; McMahon, Timothy J

    2014-12-15

    Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ~42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., "PIPA") restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ~120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo. PMID:25305182

  7. Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc): Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides.

    Science.gov (United States)

    Audia, Jonathon P; Winkler, Herbert H

    2006-09-01

    The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K(i) similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins. PMID:16923893

  8. Mitochondrial ATP synthasome: Expression and structural interaction of its components

    Czech Academy of Sciences Publication Activity Database

    Nůsková, Hana; Mráček, Tomáš; Mikulová, Tereza; Vrbacký, Marek; Kovářová, Nikola; Kovalčíková, Jana; Pecina, Petr; Houštěk, Josef

    2015-01-01

    Roč. 464, č. 3 (2015), s. 787-793. ISSN 0006-291X R&D Projects: GA ČR(CZ) GAP303/12/1363; GA MŠk(CZ) LL1204 Grant ostatní: GA UK(CZ) 1160214 Institutional support: RVO:67985823 Keywords : mitochondria * oxidative phosphorylation * supercomplexes * ATP synthasome * ATP synthase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.297, year: 2014

  9. Connexins regulate calcium signaling by controlling ATP release

    OpenAIRE

    Cotrina, Maria Luisa; Lin, Jane H.-C.; Alves-Rodrigues, Alexandra; Liu, Shujun; Li, Jiang; Azmi-Ghadimi, Hooman; Kang, Jian; Naus, Christian C.G.; Nedergaard, Maiken

    1998-01-01

    Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca2+ mobilization and was inhibited by Cl− channel blockers. Calcium wave propagation also was reduced by purinergic receptor antag...

  10. Effects of Dopamine D2 Receptor Partial Agonist Antipsychotic Aripiprazole on Dopamine Synthesis in Human Brain Measured by PET with L-[β-11C]DOPA

    OpenAIRE

    Ito, Hiroshi; Takano, Harumasa; Arakawa, Ryosuke; Takahashi, Hidehiko; Kodaka, Fumitoshi; Takahata, Keisuke; Nogami, Tsuyoshi; Suzuki, Masayuki; Suhara, Tetsuya

    2012-01-01

    Dopamine D2 receptor partial agonist antipsychotic drugs can modulate dopaminergic neurotransmission as functional agonists or functional antagonists. The effects of antipsychotics on presynaptic dopaminergic functions, such as dopamine synthesis capacity, might also be related to their therapeutic efficacy. Positron emission tomography (PET) was used to examine the effects of the partial agonist antipsychotic drug aripiprazole on presynaptic dopamine synthesis in relation to dopamine D2 rece...

  11. Sex Differences of Brain Serotonin Synthesis in Patients with Irritable Bowel Syndrome Using α-[11C]methyl-L-tryptophan, Positron Emission Tomography and Statistical Parametric Mapping

    OpenAIRE

    NAKAI, Akio; Kumakura, Yoshikata; Boivin, Michel; Rosa, Pedro; Diksic, Mirko; D’Souza, Doreen; Kersey, Kathryn

    2003-01-01

    BACKGROUND: Irritable bowel syndrome (IBS) is the most common functional bowel disorder and has a strong predominance in women. Recent data suggest that the brain may play an important role in the pathophysiology of IBS in the brain-gut axis. It is strongly suspected that serotonin (5-HT), a neurotransmitter found in the brain and gut, may be related to the pathophysiology of IBS. It is reported that a 5-HT3 antagonist is effective only in female patients with diarrhea-predominant IBS.OBJECTI...

  12. The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

    Science.gov (United States)

    Malyan, Alexander N

    2016-05-01

    The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water. PMID:26754050

  13. Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 e subunit

    Czech Academy of Sciences Publication Activity Database

    Mayr, J. A.; Havlíčková, Vendula; Zimmermann, F.; Magler, I.; Kaplanová, Vilma; Ješina, Pavel; Pecinová, Alena; Nůsková, Hana; Koch, J.; Sperl, W.; Houštěk, Josef

    2010-01-01

    Roč. 19, č. 17 (2010), s. 3430-3439. ISSN 0964-6906 R&D Projects: GA MZd(CZ) NS9759; GA MŠk(CZ) 1M0520 Grant ostatní: Univerzita Karlova(CZ) 97807 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATP-synthase * ATP5E * disease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.058, year: 2010

  14. Molecular mechanism of sulphonylurea block of K(ATP) channels carrying mutations that impair ATP inhibition and cause neonatal diabetes.

    Science.gov (United States)

    Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

    2013-11-01

    Sulphonylurea drugs are the therapy of choice for treating neonatal diabetes (ND) caused by mutations in the ATP-sensitive K(+) channel (KATP channel). We investigated the interactions between MgATP, MgADP, and the sulphonylurea gliclazide with KATP channels expressed in Xenopus oocytes. In the absence of MgATP, gliclazide block was similar for wild-type channels and those carrying the Kir6.2 ND mutations R210C, G334D, I296L, and V59M. Gliclazide abolished the stimulatory effect of MgATP on all channels. Conversely, high MgATP concentrations reduced the gliclazide concentration, producing a half-maximal block of G334D and R201C channels and suggesting a mutual antagonism between nucleotide and gliclazide binding. The maximal extent of high-affinity gliclazide block of wild-type channels was increased by MgATP, but this effect was smaller for ND channels; channels that were least sensitive to ATP inhibition showed the smallest increase in sulphonylurea block. Consequently, G334D and I296L channels were not fully blocked, even at physiological MgATP concentrations (1 mmol/L). Glibenclamide block was also reduced in β-cells expressing Kir6.2-V59M channels. These data help to explain why patients with some mutations (e.g., G334D, I296L) are insensitive to sulphonylurea therapy, why higher drug concentrations are needed to treat ND than type 2 diabetes, and why patients with severe ND mutations are less prone to drug-induced hypoglycemia. PMID:23835339

  15. Brain Tumors

    Science.gov (United States)

    A brain tumor is a growth of abnormal cells in the tissues of the brain. Brain tumors can be benign, with no cancer cells, or ... cancer cells that grow quickly. Some are primary brain tumors, which start in the brain. Others are metastatic, ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... Basics will introduce you to some of this science, such as: How the brain develops How genes and the environment affect the brain The basic structure of the brain How different parts of the brain communicate and work with each other How changes in the brain ...

  17. Brain Tumors

    Science.gov (United States)

    A brain tumor is a growth of abnormal cells in the tissues of the brain. Brain tumors can be benign, with no cancer cells, ... cancer cells that grow quickly. Some are primary brain tumors, which start in the brain. Others are ...

  18. Brain Fingerprinting

    Directory of Open Access Journals (Sweden)

    ravi kumar

    2012-12-01

    Full Text Available Brain Fingerprinting is a scientific technique to determine whether or not specific information is stored in an individual's brain by measuring a electrical brain wave response to Word, phrases, or picture that are presented on computer screen. Brain Fingerprinting is a controversial forensic science technique that uses electroencephalograph y (EEG to determine whether specific information is stored in a subject's brain

  19. Astrocytes protect neurons against methylmercury via ATP/P2Y(1 receptor-mediated pathways in astrocytes.

    Directory of Open Access Journals (Sweden)

    Yusuke Noguchi

    Full Text Available 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.

  20. Comparison of methods for specific depletion of ATP in Salmonella typhimurium.

    OpenAIRE

    Johnson, M. S.; Taylor, B L

    1993-01-01

    Three methods of ATP depletion in Salmonella typhimurium were compared. ATP concentrations were lowest after arsenate treatment. Arsenate or alpha-methylglucoside-plus-azide treatment nonspecifically lowered all nucleotide triphosphate levels. Histidine starvation in a hisF mutant was relatively specific for ATP depletion and therefore has potential in distinguishing ATP-dependent processes from processes dependent on other nucleotides.

  1. Dysregulation of select ATP-dependent chromatin remodeling factors in high trait anxiety.

    Science.gov (United States)

    Wille, Alexandra; Amort, Thomas; Singewald, Nicolas; Sartori, Simone B; Lusser, Alexandra

    2016-09-15

    Enhanced anxiety is a salient feature of a number of psychiatric disorders including anxiety disorders, trauma-related disorders and depression. Although aberrant expression of various genes has been detected in patients suffering from persistent high anxiety as well as in high anxiety rodent models, the molecular mechanisms responsible for altered transcription regulation have been poorly addressed. Transcription regulation intimately involves the contribution of chromatin modifying processes, such as histone modification and ATP-dependent chromatin remodeling, yet their role in pathological anxiety is not known. Here, we investigated for the first time if altered levels of several ATP-dependent chromatin remodeling factors (ChRFs) and histone deacetylases (HDACs) may be linked to high trait anxiety in mice. While we found protein levels of the ChRFs SNF2H, ATRX, CHD1, CHD3 and CHD5 and of HDACs 1-3 and 6 to be similar in most of the tested brain areas of mice with high (HAB) versus normal (NAB) anxiety-related behavior, we observed distinctly altered regulation of SNF2H in the amygdala, and of CHD3 and CHD5 in the ventral hippocampus. In particular, CHD3 and CHD5 exhibited altered expression of protein but not of mRNA in HAB mice. Since both proteins are components of NuRD-like complexes, these results may indicate an impaired equilibrium between different NuRD-like complexes in the ventral hippocampus. Overall, our data provide novel evidence for localized differences of specific ATP-dependent chromatin remodeling factors in mice with high trait anxiety that may ultimately contribute to altered transcriptional programs resulting in the manifestation of pathological anxiety. PMID:27208790

  2. Oligomycin frames a common drug-binding site in the ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

    2015-12-01

    We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

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

    OpenAIRE

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

    2014-01-01

    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 c...

  4. Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics.

    OpenAIRE

    Naito, Tomoki; Takatsu, Hiroyuki; Miyano, Rie; Takada, Naoto; Nakayama, Kazuhisa; Shin, Hye-Won

    2015-01-01

    We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543-33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of the ... distant nerve cells (via axons) to form brain circuits. These circuits control specific body functions such as ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... Brain Basics provides information on how the brain works, how mental illnesses are disorders of the brain, ... others live with symptoms of mental illness every day. They can be moderate, or serious and cause ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... helps Sarah to better cope with her feelings. Brain Research Modern research tools and techniques are giving scientists ... the treatment for a person's specific conditions. Such brain research help increase the understanding of how the brain ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... little dopamine or problems using dopamine in the thinking and feeling regions of the brain may play ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of the brain ... specialized for the function of conducting messages. A neuron has three basic parts: Cell body which includes ...

  11. Brain Malformations

    Science.gov (United States)

    Most brain malformations begin long before a baby is born. Something damages the developing nervous system or causes it ... medicines, infections, or radiation during pregnancy interferes with brain development. Parts of the brain may be missing, ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... brain's structure, studies show that brain growth in children with autism appears to peak early. And as ... grow there are differences in brain development in children who develop bipolar disorder than children who do ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... Basics will introduce you to some of this science, such as: How the brain develops How genes and the environment affect the brain The basic structure of the brain How different parts of ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... understanding of the brain than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... science, such as: How the brain develops How genes and the environment affect the brain The basic ... that with brain development in people mental disorders. Genes and environmental cues both help to direct this ...

  16. Brain surgery

    Science.gov (United States)

    Craniotomy; Surgery - brain; Neurosurgery; Craniectomy; Stereotactic craniotomy; Stereotactic brain biopsy; Endoscopic craniotomy ... cut depends on where the problem in the brain is located. The surgeon creates a hole in ...

  17. 新型苯并硫氮杂(卓)酮类非ATP竞争GSK-3β抑制剂的设计、合成和活性评价%Design, Synthesis and in Vitro Test of Novel Non-ATP Competitive Glycogen Synthase Kinase-3β(GSK-3β)Inhibitors

    Institute of Scientific and Technical Information of China (English)

    黄朝辉; 胡海荣; 雷贾毅; 楚勇; 叶德泳

    2012-01-01

    OBJECTIVE To discover novel non-ATP competitive glycogen synthase kinase-3P(GSK-3P) inhibitors. METHODS A virtual screening was conducted by Autodock program, which docked the small drug-like molecules of Maybridge library at the non-ATP binding site of GSK-3β The target compounds had been designed based on the virtual screening result and successfully synthesized through Knoevenagel reaction, cyclization and Af-alkylation. The inhibition to GSK-3P was tested by in vitro enzamic test. RESULTS 5-benzyl-2-(furan-2-yl)-2,3-dihydrobenzo[b][l,4] thiazepin-4(5H)-one showed moderate inhibition to GSK-3P in vitro (IC50 47.69±2.38 μmol·L-1). CONCLUSION The discovered new active compound is structurally different to other inhibitors of GSK-3P and worthy of further study as a novel lead compound.%目的 寻找新型的非ATP竞争糖原合成酶激酶-3β(GSK-3β)抑制剂.方法 针对GSK-3β的非ATP结合的底物作用位点为靶点,采用Autodock程序对类药性小分子库Maybridge进行虚拟筛选寻找新型GSK-3β抑制剂.采用克脑文格尔反应,环合及N-烷基化反应制备目标化合物.采用体外酶抑制活性测试目标化合物的活性.结果 化合物2-(2-呋喃基)-5-苄基-2,3-二氢苯并[b][1,4]硫氮杂(卓)-4(5H)-酮对GSK-3β具有中等抑制活性(IC50 47.69±2.38 μmol·L-1).结论 活性化合物的结构与目前报道的其他GSK-3β抑制剂不同,可望作为新的先导化合物,值得进一步研究.

  18. Structure of the nucleotide-binding subunit B of the energy producer A1A0 ATP synthase in complex with adenosine diphosphate.

    Science.gov (United States)

    Kumar, Anil; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

    2008-11-01

    A1A0 ATP synthases are the major energy producers in archaea. Like the related prokaryotic and eukaryotic F1F0 ATP synthases, they are responsible for most of the synthesis of adenosine triphosphate. The catalytic events of A1A0 ATP synthases take place inside the A3B3 hexamer of the A1 domain. Recently, the crystallographic structure of the nucleotide-free subunit B of Methanosarcina mazei Gö1 A1A0 ATP synthase has been determined at 1.5 A resolution. To understand more about the nucleotide-binding mechanism, a protocol has been developed to crystallize the subunit B-ADP complex. The crystallographic structure of this complex has been solved at 2.7 A resolution. The ADP occupies a position between the essential phosphate-binding loop and amino-acid residue Phe149, which are involved in the binding of the antibiotic efrapeptin in the related F1F0 ATP synthases. This trapped ADP location is about 13 A distant from its final binding site and is therefore called the transition ADP-binding position. In the trapped ADP position the structure of subunit B adopts a different conformation, mainly in its C-terminal domain and also in the final nucleotide-binding site of the central alphabeta-domain. This atomic model provides insight into how the substrate enters into the nucleotide-binding protein and thereby into the catalytic A3B3 domain. PMID:19020348

  19. Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones.

    Science.gov (United States)

    Johansson, Renzo; Jonna, Venkateswara Rao; Kumar, Rohit; Nayeri, Niloofar; Lundin, Daniel; Sjöberg, Britt-Marie; Hofer, Anders; Logan, Derek T

    2016-06-01

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone. PMID:27133024

  20. Brain mapping

    OpenAIRE

    Blaž Koritnik

    2004-01-01

    Cartography of the brain ("brain mapping") aims to represent the complexities of the working brain in an understandable and usable way. There are four crucial steps in brain mapping: (1) acquiring data about brain structure and function, (2) transformation of data into a common reference, (3) visualization and interpretation of results, and (4) databasing and archiving. Electrophysiological and functional imaging methods provide information about function of the human brain. A prere...

  1. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

    Science.gov (United States)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1981-01-01

    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  2. Characterization of Na+ influx mediated by ATP(4-)-activated P2 purinoceptors in PC12 cells.

    OpenAIRE

    Choi, S. Y.; Kim, K. T.

    1996-01-01

    1. Micromolar levels of extracellular ATP increased cytosolic Na+ concentration ([Na+]i) as well as cytosolic Ca2+ concentration ([Ca2+]i) in PC12 cells. 2. Pretreatment of cells with tetrodotoxin, benzamil or thapsigargin did not alter the ATP-induced Na+ influx. 3. Increased extracellular Mg2+ concentration decreased the ATP effect. Furthermore, when the extracellular ATP pool was treated to contain corresponding calculated concentrations of ATP4-, the increase in [Na+]i stayed linked to th...

  3. ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding

    OpenAIRE

    Liu, Fei; Putnam, Andrea; Jankowsky, Eckhard

    2008-01-01

    DEAD-box proteins, the largest helicase family, catalyze ATP-dependent remodeling of RNA–protein complexes and the unwinding of RNA duplexes. Because DEAD-box proteins hydrolyze ATP in an RNA-dependent fashion, the energy provided by ATP hydrolysis is commonly assumed to drive the energetically unfavorable duplex unwinding. Here, we show efficient unwinding of stable duplexes by several DEAD-box proteins in the presence of the nonhydrolyzable ATP analog ADP-beryllium fluoride. Another ATP ana...

  4. The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria.

    Science.gov (United States)

    Macchioni, Lara; Davidescu, Magdalena; Roberti, Rita; Corazzi, Lanfranco

    2014-10-01

    Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H(2)O(2) release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells. PMID:25194986

  5. 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*

    OpenAIRE

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

    2013-01-01

    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 ⇆ 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, ...

  6. Rotation and structure of FoF1-ATP synthase.

    Science.gov (United States)

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2011-06-01

    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

  7. Phenomenological analysis of ATP dependence of motor protein

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, we found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motor can be well approximated by a Michaelis-Menten like formula $V=\\atp k(F)L/(\\atp +K_M)$, with $L$ the step size, and $k(F)$ the external load $F$ dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant $K_M$ for substall, superstall and negative external load indicates, the ATP molecule affinity of motor head for these three cases are different, though the expression of $k(F)$ as a function of $F$ might be unchanged for any external load $F$. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

  8. Brain and heart disease studies

    International Nuclear Information System (INIS)

    Highlights of important studies completed during the past year using the Donner 280-crystal positron ring tomograph are summarized in this article. Using rubidium-82, images of a brain tumor and an arteriovenous malformation are described. An image demonstrating methionine uptake in a patient with schizophrenia and an image reflecting sugar metabolism in the brain of a man with Alzheimer's disease are also included. Uptake of rubidium-82 in subjects before and after exercise is being investigated. The synthesis of new radiopharmaceuticals and the development of a new synthesis for C-taurine for use in the study of metabolism in the human heart are also being studied

  9. [Neurochemical study of effects of the new anxiolytic drugs afobazol and ladasten on the synthesis and metabolism of monoamines and their metabolites in the brain structures of Wistar rat on the model of monoamine synthesis blockade induced by aromatic amino acid decarboxylase inhibitor NSD-1015].

    Science.gov (United States)

    Davydova, A I; Klodt, P M; Kudrin, V S; Kuznetsova, E A; Narkevich, V B

    2010-03-01

    Results of a neurochemical study of the effects of the new anxiolytic drugs afobazole and ladasten on the synthesis and metabolism of monoamines and their metabolites determined by HPLC on the model of monoamine synthesis blockade induced by NSD-1015 (aromatic L-amino acid decarboxylase) in the brain structures of Wistar rats are reported. A decrease in the levels of DOPAC in hypothalamus and HVA in striatum after afobazole injection may be evidence of an inhibitory action of this drug on the activity of monoamine oxidase (MAO-A), which is the main enzyme involved in dopamine biodegradation. Afobazole was also found to increase the content of serotonin (5-HT) as well as its precursor (5-OTP) and its main metabolite (5-HIAA) in hypothalamus by up to 50, 60 and 50%, respectively, which confirms a hypothesis that this anxiolytic drug can modulate the activity of tryptophan hydroxylase (5-OTP synthesis enzyme). In contrast to afobazole, ladasten demonstrated the ability to increase the level of L-DOPA (a dopamine precursor) in virtually all functional structures of the brain (except for hippocamp), which may support the hypothesis suggestion concerning a predominant action of this drug on the activity of tyrosine hydroxylase. Ladasten exhibited selectivity with respect to the dopaminergic system and affected only parameters of the dopamine metabolism, in particular, by increasing the HVA content in nucleus accumbens and decreasing it in the hypothalamus. The drug also affected the dopamine turnover parameters, producing an increase in both HVA/dopamine ratio in nucleus accumbens and DOPAC/dopamine ratio in hippocamp. PMID:20408420

  10. Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly

    OpenAIRE

    Csanadi, Agnes; Kayser, Claudia; Donauer, Marcel; Gumpp, Vera; Aumann, Konrad; Rawluk, Justyna; Prasse, Antje; zur Hausen, Axel; Wiesemann, Sebastian; Werner, Martin; Kayser, Gian

    2015-01-01

    Background Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient’s prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules ‘(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biologica...

  11. The Buecherer-Strecker synthesis of D- and L-(1-11C)tyrosine and the in vivo study of 0100L-(1-11C)tyrosine in human brain using positron emission tomography

    International Nuclear Information System (INIS)

    The synthesis of D- and L-(1-11C)tyrosine, starting with 11C-cyanide, is reported. DL-(1-11C)tyrosine was prepared by the Buecherer-Strecker reaction, from carrier added 11C-cyanide with an incorporation of 80% in 20 min. The isolation of the pure D- and L-amino acid isomers from the enantiomeric mixture was accomplished within 15 min by preparative HPLC using a chiral stationary phase and a phosphate buffer as the mobile phase. Typically, the total synthesis time was 50 min (including purification) from end of trapping of 11C-cyanide, with a radiochemical yield of D- and L-amino acid of 40%-60%. The D- and L-(1-11C)tyrosine were both obtained optically pure, with a carrier added specific activity of 0.3-0.5 Ci/mmol and a radiochemical purity better than 99%. The 11C labelled L-tyrosine was used in an in vivo study in the human brain using positron emission tomography (PET). (orig.)

  12. Synthesis of radiotracers for studying muscarinic cholinergic receptors in the living human brain using positron emission tomography: [11C]dexetimide and [11C]levetimide

    International Nuclear Information System (INIS)

    The localization and quantitation of muscarinic cholinergic receptors (m-AChR) in the living human brain using a non-invasive method such as positron emission tomography (PET) may provide valuable information about receptor changes which have been observed post mortem in patients with Huntington's chorea and Alzheimer's dementia, as well as normal brain mechanisms mediated by the m-AChR. We chose to label dexetimide as a radiotracer for studying the m-AChR and levetimide as a radiotracer for assessing non-specific binding associated with the in vivo receptor binding studies. (author)

  13. Sperm motility and ATP content in seminal hyperviscosity.

    Science.gov (United States)

    Mendeluk, G R; Munuce, M J; Carizza, C; Sardi, M; Bregni, C

    1997-01-01

    Objective spermatic motility (Hamilton Thorne Research), the rapid progressive spermatozoa (grade A) recovery after swim-up, and the spermatozoa ATP content (bioluminescence) were studied in normoviscous and hyperviscous asthenospermic samples. The amplitude of lateral head displacement (ALH) was significantly lower in hyperviscous semen (normal: 4.6 +/- 0.7 microns [n = 20], high: 3.5 +/- 1.2 microns [n = 16]; p semens with high consistency (normal: 71.0 +/- 38.0 [n = 14], high: 181.3 +/- 108.9 [n = 6]; p < .05). The ATP content per living spermatozoa was in the normal consistency group 449.4 +/- 65.1 pmol per million living spermatozoa (n = 29) and in the high consistency batch 605.1 +/- 242.8 (n = 9), p < .05. In asthenospermia, the spermatozoa from hyperviscous samples have minor ALH values, better response to swim-up, and high ATP content than those from normoviscous ejaculates. PMID:9352034

  14. The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

    Science.gov (United States)

    Goswami, Srikanta; Adhya, Samit

    2006-07-14

    Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import. PMID:16735512

  15. Brain Basics

    Medline Plus

    Full Text Available ... in Real Life Brain Research Glossary Brain Basics (PDF, 10 pages) Introduction Watch the Brain Basics video ... early brain development, and may also assist in learning and memory. ... rise to disabilities or diseases. neural circuit —A network of neurons ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies show that brain growth in children with autism appears to peak early. And as ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... Research Modern research tools and techniques are giving scientists a more detailed understanding of the brain than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... Welcome. Brain Basics provides information on how the brain works, how mental illnesses are disorders of the brain, ... highly developed area at the front of the brain that, in humans, plays a role in executive functions such as ...

  19. ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols.

    Science.gov (United States)

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2016-02-01

    ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions. PMID:26589785

  20. Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy

    CERN Document Server

    Renz, Marc; Boersch, Michael

    2012-01-01

    FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal proton-driven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic hypothesis', a series of proton pumps generate the necessary pH difference plus an electric potential across the bacterial plasma membrane. These proton pumps are redox-coupled membrane enzymes which are possibly organized in supercomplexes, as shown for the related enzymes in the mitochondrial inner membrane. We report diffusion measurements of single fluorescent FoF1-ATP synthases in living E. coli by localization microscopy and single enzyme tracking to distinguish a monomeric enzyme from a supercomplex-associated form in the bacterial membrane. For quantitative mean square displacement (MSD) analysis, the limited size of the observation area in the membrane with a significant membrane curvature had to be considered. The E. coli cells had a diameter of about 500 nm and a length o...

  1. Dimethylfumarate inhibits microglial and astrocytic inflammation by suppressing the synthesis of nitric oxide, IL-1β, TNF-α and IL-6 in an in-vitro model of brain inflammation

    Directory of Open Access Journals (Sweden)

    Mrowietz Ulrich

    2010-05-01

    Full Text Available Abstract Background Brain inflammation plays a central role in multiple sclerosis (MS. Dimethylfumarate (DMF, the main ingredient of an oral formulation of fumaric acid esters with proven therapeutic efficacy in psoriasis, has recently been found to ameliorate the course of relapsing-remitting MS. Glial cells are the effector cells of neuroinflammation; however, little is known of the effect of DMF on microglia and astrocytes. The purpose of this study was to use an established in vitro model of brain inflammation to determine if DMF modulates the release of neurotoxic molecules from microglia and astrocytes, thus inhibiting glial inflammation. Methods Primary microglial and astrocytic cell cultures were prepared from cerebral cortices of neonatal rats. The control cells were treated with LPS, an accepted inducer of pro-inflammatory properties in glial cells, and the experimental groups with LPS and DMF in different concentrations. After stimulation/incubation, the generation of nitric oxide (NO in the cell culture supernatants was determined by measuring nitrite accumulation in the medium using Griess reagent. After 6 hours of treatment RT-PCR was used to determine transcription levels of iNOS, IL-1β, IL-6 and TNF-α mRNA in microglial and astrocytic cell cultures initially treated with DMF, followed after 30 min by LPS treatment. Moreover, we investigated possible involvement of the ERK and Nrf-2 transduction pathway in microglia using western blot analysis. Results Pretreatment with DMF decreased synthesis of the proinflammatory mediators iNOS, TNF-α, IL-1β and IL-6 at the RNA level in activated microglia and astrocytes in vitro, associated with a decrease in ERK phosphorylation in microglia. Conclusions Collectively, these results suggest that the neuroprotective effects of DMF may be in part functionally attributable to the compound's ability to inhibit expression of multiple neuroinflammatory mediators in brain of MS patients.

  2. Authentic role of ATP signaling in micturition reflex

    OpenAIRE

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-01

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder f...

  3. Structure and mechanism of ATP-dependent phospholipid transporters

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Poulsen, Lisbeth Rosager; Bailly, Aurélien;

    2015-01-01

    Background ATP-binding cassette (ABC) transporters and P4-ATPases are two large and seemingly unrelated families of primary active pumps involved in moving phospholipids from one leaflet of a biological membrane to the other. Scope of review This review aims to identify common mechanistic features...... in the way phospholipid flipping is carried out by two evolutionarily unrelated families of transporters. Major conclusions Both protein families hydrolyze ATP, although they employ different mechanisms to use it, and have a comparable size with twelve transmembrane segments in the functional unit...

  4. Mechanism of decline in rat brain 5-hydroxytryptamine after induction of liver tryptophan pyrrolase by hydrocortisone: roles of tryptophan catabolism and kynurenine synthesis

    OpenAIRE

    Young, Simon N.

    1981-01-01

    1 Two mechanisms have been proposed to explain the decline in brain tryptophan and 5-hydroxytryptamine (5-HT) after administration of hydrocortisone and the subsequent induction of liver pyrrolase. These are depletion of tryptophan by high rates of tryptophan catabolism and inhibition of tryptophan uptake by elevated levels of the tryptophan catabolite, kynurenine.

  5. Brain mapping

    Directory of Open Access Journals (Sweden)

    Blaž Koritnik

    2004-08-01

    Full Text Available Cartography of the brain ("brain mapping" aims to represent the complexities of the working brain in an understandable and usable way. There are four crucial steps in brain mapping: (1 acquiring data about brain structure and function, (2 transformation of data into a common reference, (3 visualization and interpretation of results, and (4 databasing and archiving. Electrophysiological and functional imaging methods provide information about function of the human brain. A prerequisite for multisubject, multidimensional and multimodal mapping is transformation of individual images to match a standard brain template. To produce brain maps, color, contours, and other visual cues are used to differentiate metabolic rates, electrical field potentials, receptor densities, and other attributes of structure or function. Databases are used to organize and archive data records. By relating the maps to cognitive functions and psychological models, brain mapping offers a prerequisite for the understanding of organizational principles of the human brain.

  6. Conjugates of Superoxide Dismutase 1 with Amphiphilic Poly(2-oxazoline) Block Copolymers for Enhanced Brain Delivery: Synthesis, Characterization and Evaluation in Vitro and in Vivo

    KAUST Repository

    Tong, Jing

    2013-01-07

    Superoxide dismutase 1 (SOD1) efficiently catalyzes dismutation of superoxide, but its poor delivery to the target sites in the body, such as brain, hinders its use as a therapeutic agent for superoxide-associated disorders. Here to enhance the delivery of SOD1 across the blood-brain barrier (BBB) and in neurons the enzyme was conjugated with poly(2-oxazoline) (POx) block copolymers, P(MeOx-b-BuOx) or P(EtOx-b-BuOx), composed of (1) hydrophilic 2-methyl-2-oxazoline (MeOx) or 2-ethyl-2-oxazoline (EtOx) and (2) hydrophobic 2-butyl-2-oxazoline (BuOx) repeating units. The conjugates contained from 2 to 3 POx chains joining the protein amino groups via cleavable -(ss)- or noncleavable -(cc)- linkers at the BuOx block terminus. They retained 30% to 50% of initial SOD1 activity, were conformationally and thermally stable, and assembled in 8 or 20 nm aggregates in aqueous solution. They had little if any toxicity to CATH.a neurons and displayed enhanced uptake in these neurons as compared to native or PEGylated SOD1. Of the two conjugates, SOD1-(cc)-P(MeOx-b-BuOx) and SOD1-(cc)-P(EtOx-b-BuOx), compared, the latter was entering cells 4 to 7 times faster and at 6 h colocalized predominantly with endoplasmic reticulum (41 ± 3%) and mitochondria (21 ± 2%). Colocalization with endocytosis markers and pathway inhibition assays suggested that it was internalized through lipid raft/caveolae, also employed by the P(EtOx-b-BuOx) copolymer. The SOD activity in cell lysates and ability to attenuate angiotensin II (Ang II)-induced superoxide in live cells were increased for this conjugate compared to SOD1 and PEG-SOD1. Studies in mice showed that SOD1-POx had ca. 1.75 times longer half-life in blood than native SOD1 (28.4 vs 15.9 min) and after iv administration penetrated the BBB significantly faster than albumin to accumulate in brain parenchyma. The conjugate maintained high stability both in serum and in brain (77% vs 84% at 1 h postinjection). Its amount taken up by the brain

  7. α3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish

    DEFF Research Database (Denmark)

    Doganli, Canan; Beck, Hans Christian; Ribera, Angeles B;

    2013-01-01

    Na+/K+-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na+/K+-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder...... knockdown of Atp1a3a or Atp1a3b. Our data thus strongly support the role of α3Na+/K+-ATPase in zebrafish motility and brain development, associating for the first time the α3Na+/K+-ATPase deficiency with brain ventricle dilation....

  8. Effects of Irradiation on bacterial atp luminous intensity of cooled pork and chicken

    International Nuclear Information System (INIS)

    The effect of irradiation on cooled pork and chicken was detected with ATP luminous intensity method. The influences of other factors to ATP luminous intensity were also discussed. There was positive correlation between ATP standard concentration and ATP luminous intensity, and negative correlation between irradiation dosage and ATP luminous intensity. The trend of ATP luminous intensity of cooled pork and chicken after irradiation was inverse S, and the maximum ATP luminous intensity appeared at 6.0 kGy, and minimum at 4.0 and 8.0 kGy. Sterilized water and sterilized pork had no interference to ATP luminous intensity of the samples. There was significant positive correlation between E. coli 10003 concentration and ATP luminous intensity, the coefficient correlation was 0.9437. (authors)

  9. Synthesis, 18F-Radiolabelling and Biological Characterization of Novel Fluoroalkylated Triazine Derivatives for in Vivo Imaging of Phosphodiesterase 2A in Brain via Positron Emission Tomography

    Directory of Open Access Journals (Sweden)

    Susann Schröder

    2015-05-01

    Full Text Available Phosphodiesterase 2A (PDE2A is highly and specifically expressed in particular brain regions that are affected by neurological disorders and in certain tumors. Development of a specific PDE2A radioligand would enable molecular imaging of the PDE2A protein via positron emission tomography (PET. Herein we report on the syntheses of three novel fluoroalkylated triazine derivatives (TA2–4 and on the evaluation of their effect on the enzymatic activity of human PDE2A. The most potent PDE2A inhibitors were 18F-radiolabelled ([18F]TA3 and [18F]TA4 and investigated regarding their potential as PET radioligands for imaging of PDE2A in mouse brain. In vitro autoradiography on rat brain displayed region-specific distribution of [18F]TA3 and [18F]TA4, which is consistent with the expression pattern of PDE2A protein. Metabolism studies of both [18F]TA3 and [18F]TA4 in mice showed a significant accumulation of two major radiometabolites of each radioligand in brain as investigated by micellar radio-chromatography. Small-animal PET/MR studies in mice using [18F]TA3 revealed a constantly increasing uptake of activity in the non-target region cerebellum, which may be caused by the accumulation of brain penetrating radiometabolites. Hence, [18F]TA3 and [18F]TA4 are exclusively suitable for in vitro investigation of PDE2A. Nevertheless, further structural modification of these promising radioligands might result in metabolically stable derivatives.

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

    Science.gov (United States)

    Jaramillo, Michael L; Abanto, Michel; Quispe, Ruth L; Calderón, Julio; Del Valle, Luís J; Talledo, Miguel; Ramírez, Pablo

    2012-01-01

    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. PMID:23055613

  11. EFFECTS OF CONTINUOUS-WAVE, PULSED, AND SINUSOIDAL-AMPLITUDE-MODULATED MICROWAVES ON BRAIN ENERGY METABOLISM

    Science.gov (United States)

    A comparison of the effects of continuous wave, sinusoidal-amplitude modulated, and pulsed square-wave-modulated 591-MHz microwave exposures on brain energy metabolism was made in male Sprague Dawley rats (175-225g). Brain NADH fluorescence, adensine triphosphate (ATP) concentrat...

  12. Elucidation of the ATP7B N-domain Mg2+-ATP coordination site and its allosteric regulation.

    Directory of Open Access Journals (Sweden)

    Claude Hercend

    Full Text Available 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 Mg(2+-ATP coordination site and answer to the controversial role of the Mg(2+ 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 Mg(2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process.

  13. K-ATP channel expression and pharmacological in vivo and in vitro studies of the K-ATP channel blocker PNU-37883A in rat middle meningeal arteries

    DEFF Research Database (Denmark)

    Ploug, K.B.; Boni, L.J.; Baun, M.;

    2008-01-01

    Background and purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K+ (K-ATP) channel opening may cause migraine by dilatating...... intracranial arteries, including the middle meningeal artery (MMA). We studied the K-ATP channel expression profile in rat MMA and examined the potential inhibitory effects of the K-ATP channel blocker PNU-37883A on K-ATP channel opener-induced relaxation of the rat MMA, using the three K-ATP channel openers...... levcromakalim, pinacidil and P-1075. Experimental approach: mRNA and protein expression of K-ATP channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K-ATP channel drugs on rat MMA were studied in the genuine...

  14. Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line.

    Science.gov (United States)

    Konopacka, Agnieszka; Zielińska, Magdalena; Albrecht, Jan

    2008-05-01

    Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy. PMID:18222015

  15. Effects of nucleotides on ATP-dependent protein translocation into Escherichia coli membrane vesicles.

    OpenAIRE

    Chen, L.; Tai, P C

    1986-01-01

    We have shown previously that Escherichia coli can translocate the same protein either co- or posttranslationally and that ATP hydrolysis is essential for the posttranslational translocation of the precursors of alkaline phosphatase and OmpA protein into inverted E. coli membrane vesicles. ATP-dependent protein translocation has now been further characterized. In the absence of exogenous Mg2+, dATP, formycin A-5'-triphosphate, ATP-alpha-S, and N1-oxide-ATP could replace ATP, but many other nu...

  16. Carbon and energy metabolism of atp mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    The membrane-bound H+-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth...

  17. [Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP].

    Science.gov (United States)

    Emel'ianov, V V

    2007-01-01

    One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated. PMID:17380892

  18. Motor pathway excitability in ATP13A2 mutation carriers

    DEFF Research Database (Denmark)

    Zittel, S; Kroeger, J; van der Vegt, J P M;

    2012-01-01

    OBJECTIVE: To describe excitability of motor pathways in Kufor-Rakeb syndrome (PARK9), an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration caused by a mutation in the ATP13A2 gene, using transcranial magnetic stimulation (TMS). METHODS: Five members of a Chilean family with...

  19. Detection of ATP and NADH: A Bioluminescent Experience.

    Science.gov (United States)

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  20. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  1. ATP economy of force maintenance in human tibialis anterior muscle

    DEFF Research Database (Denmark)

    Nakagawa, Yoshinao; Ratkevicius, Aivaras; Mizuno, Masao;

    2005-01-01

    PURPOSE: The aim of this study was investigate ATP economy of force maintenance in the human tibialis anterior muscle during 60 s of anaerobic voluntary contraction at 50% of maximum voluntary contraction (MVC). METHODS: ATP turnover rate was evaluated using P magnetic resonance spectroscopy (P...... +/- SEM) of the total ankle dorsiflexor muscle volume, which was 267 +/- 10 cm. Relative cross-sectional areas occupied by Type I, IIA, and IIB fibers in the tibialis anterior were 69.3 +/- 2.2, 27.4 +/- 2.76, and 3.2 +/- 1.0%, respectively. ATP economy of force maintenance did not change significantly...... during the 60-s contraction. It averaged at 4.81 +/- 0.42 N.s.micromol-1, and correlated with the relative cross-sectional area of the muscle occupied by Type I fiber (r = 0.73, P < 0.01). For the second half of the contraction, subjects dropping in force showed lower ATP economy compared with those...

  2. Teacher Development Program for ATP 2000. Project Report.

    Science.gov (United States)

    Sutphin, Dean; And Others

    Agri Tech Prep 2000 (ATP 2000) is a 4-year tech prep program linking high school and postsecondary curricula designed to prepare New York students for careers in agriculture or acceptance into a college program in agriculture. Because teacher development was designated an integral project component for fiscal year 1991-1992, a weeklong teacher…

  3. Abiogenic photophosphorylation of ADP to ATP sensitized by flavoproteinoid microspheres.

    Science.gov (United States)

    Kolesnikov, Michael P; Telegina, Taisiya A; Lyudnikova, Tamara A; Kritsky, Mikhail S

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10-20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer (F1H*) and ADP are involved. PMID:18386156

  4. ATP stimulates calcium influx in primary astrocyte cultures

    International Nuclear Information System (INIS)

    The effect of ATP and other purines on 45Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular 45Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of 45Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced 45Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

  5. Paradox applications integration ATP's for MAC and mass balance programs

    International Nuclear Information System (INIS)

    The K Basins Materials Accounting (MAC) and Material Balance (MBA) database system were set up to run under one common applications program. This Acceptance Test Plan (ATP) describes how the code was to be tested to verify its correctness. The scope of the tests is minimal, since both MAC and MBA have already been tested in detail as stand-alone programs

  6. Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

    International Nuclear Information System (INIS)

    Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of 125[I] labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation

  7. In silico analysis, mapping of regulatory elements and corresponding protein-DNA interaction in atp-beta gene promoter from different tomato varieties

    International Nuclear Information System (INIS)

    Chloroplast atp-beta gene encodes the beta-subunit of ATP synthase having a function in the synthesis of ATP. ATP synthase is usually present in the mitochondrial and chloroplast membranes as well as in prokaryotes with a highly conserved structure. With the aim to study the functional diversity, atpB gene promoter from Solanum lycopersicum varieties (VCT-1, 127, PETO-86, LBR-16, Continental, LBR-06, CLN-2498D, CLN-2777, LBR-09) was amplified, sequenced and analyzed. BLAST tool analyzed the sequences. Cis acting elements were predicted using Consite tool in the promoter region as these elements have importance in gene regulation and maps of the cis-acting elements were constructed with the help of DOG software for each variety. It was seen from the constructed maps that the distribution pattern of trans-acting elements was quite diverse in the atpB promoter of studied tomato varieties. Various trans-acting elements (HMG-IY, HFH-2, TBP, c-Fos and SOX17) were docked with respective DNA sequence using HADDOCK online software. Main focus was on the analysis of DNA-protein interactions and for this purpose; hydrogen bonds formed with either phosphate backbone or base of DNA were checked. Particularly interactions between amino acid and base pairing were checked. It was revealed that Arginine and Lysine had a greater probability to interact with Thymine and Adenine as compared to other bases and all bonds formed were feasible as their distances were less than 3.5 degree A. During the analysis of interactions, it was also found that Lysine, Arginine, Asparagine and Serine are well capable to bind with Thymine while Glycine and Lysine have made bonds mostly with Adenine. (author)

  8. Targeting brain serotonin synthesis: insights into neurodevelopmental disorders with long-term outcomes related to negative emotionality, aggression and antisocial behaviour

    OpenAIRE

    Lesch, Klaus-Peter; Araragi, Naozumi; Waider, Jonas; van den Hove, Daniel; Gutknecht, Lise

    2012-01-01

    Aggression, which comprises multi-faceted traits ranging from negative emotionality to antisocial behaviour, is influenced by an interaction of biological, psychological and social variables. Failure in social adjustment, aggressiveness and violence represent the most detrimental long-term outcome of neurodevelopmental disorders. With the exception of brain-specific tryptophan hydroxylase-2 (Tph2), which generates serotonin (5-HT) in raphe neurons, the contribution of gene variation to aggres...

  9. Synthesis and characterization of a 11C-labelled derivative of S12968: an attempt to image in vivo brain calcium channels

    International Nuclear Information System (INIS)

    [11C]S11568 (3-ethyl 5-methyl 2-[2-(2-aminoethoxy)ethoxymethyl]-4-(2,3-dichlorophenyl)-6-methyl-1,4 -dihydropyridine-3,5-dicarboxylate) is a powerful ligand for the visualization of the cardiac calcium channel in vivo using PET. The aim of the present study was to synthesize a lipophilic, nonionized derivative of S11568 to facilitate its penetration into the brain. To increase the lipophilicity and to remove simultaneously the ionic nature of our ligand, the N-tert-butoxycarbonyl (N-Boc) derivative of S11568 was synthesized. An IC50 value of 1.7 nM for this derivative confirmed that both the affinity and selectivity for the calcium channel was unaltered by this chemical modification (S11568 with IC50 value of 9.9 nM). The biologically more active enantiomer of S11568, the levogyre isomer S12968, was labelled with 11C using [11C]iodomethane. The lipophilicity of the N-Boc derivative was increased by a factor of three to four when compared to the parent compound (as determined by the measurement of the octanol/buffer partition coefficients). In vivo, this derivative slightly crosses the blood-brain barrier, as demonstrated by a 4-fold increase (with respect to the parent compound S12968) of the radioactivity in the brain using the 11C-labelled N-Boc S12968. This uptake remained too low to be suitable for imaging calcium channels

  10. Synthesis and Biological Evaluation of an 18Fluorine-Labeled COX Inhibitor—[18F]Fluorooctyl Fenbufen Amide—For Imaging of Brain Tumors

    Directory of Open Access Journals (Sweden)

    Ying-Cheng Huang

    2016-03-01

    Full Text Available Molecular imaging of brain tumors remains a great challenge, despite the advances made in imaging technology. An anti-inflammatory compound may be a useful tool for this purpose because there is evidence of inflammatory processes in brain tumor micro-environments. Fluorooctylfenbufen amide (FOFA was prepared from 8-chlorooctanol via treatment with potassium phthalimide, tosylation with Ts2O, fluorination with KF under phase transfer catalyzed conditions, deprotection using aqueous hydrazine, and coupling with fenbufen. The corresponding radiofluoro product [18F]FOFA, had a final radiochemical yield of 2.81 mCi and was prepared from activated [18F]F− (212 mCi via HPLC purification and concentration. The radiochemical purity was determined to be 99%, and the specific activity was shown to exceed 22 GBq/μmol (EOS based on decay-corrected calculations. Ex-vivo analysis of [18F]FOFA in plasma using HPLC showed that the agent had a half-life of 15 min. PET scanning showed significant accumulation of [18F]FOFA over tumor loci with reasonable contrast in C6-glioma bearing rats. These results suggest that this molecule is a promising agent for the visualization of brain tumors. Further investigations should focus on tumor micro-environments.

  11. In vivo Dynamic Studies of Brain Metabolism

    Institute of Scientific and Technical Information of China (English)

    LUO Xuechun; JIANG Yufeng; ZHANG Riqing

    2005-01-01

    Nuclear magnetic resonance (NMR) can noninvasively monitor intracellular concentrations and kinetic properties of numerous inorganic and organic compounds. A 31P NMR surface coil was used in vivo to dynamically measure phosphocreatine (PCr), adenosine triphosphate (ATP), and intracellular inorganic phosphate (Pi) levels in mouse brain during ischemia-reperfusion to study the damage of cerebral tissues caused by ischemia and effects of herbs on cerebral energy metabolism during ischemia-reperfusion. The study provides dynamic brain energy metabolism data during different periods. The data show that some herbs more rapidly increase the PCr level during the recovery phase than in the control group.

  12. Clinical spectrum of Kufor-Rakeb syndrome in the Chilean kindred with ATP13A2 mutations.

    Science.gov (United States)

    Behrens, Maria I; Brüggemann, Norbert; Chana, Pedro; Venegas, Pablo; Kägi, Marianne; Parrao, Teresa; Orellana, Patricia; Garrido, Cristian; Rojas, Cecilia V; Hauke, Jan; Hahnen, Eric; González, Rafael; Seleme, Nicolas; Fernández, Verónica; Schmidt, Alexander; Binkofski, Ferdinand; Kömpf, Detlef; Kubisch, Christian; Hagenah, Johann; Klein, Christine; Ramirez, Alfredo

    2010-09-15

    We report the clinical features of the original Chilean family with Kufor-Rakeb syndrome (KRS) that led to the discovery of the ATP13A2 gene at the PARK9 locus. KRS is a rare juvenile-onset autosomal recessive disease characterized by progressive Parkinsonism, pyramidal signs, and cognitive decline in addition to vertical gaze palsy and facial-faucial-finger minimyoclonus. Neurological and neuropsychological examination during a 10-year period, videotaping, neuroimaging, and measurement of DNA methylation of the ATP13A2 promoter region were performed. The youngest 5 of 17 children of nonconsanguineous parents, carrying compound-heterozygous ATP13A2 mutations, had normal development until ages ∼10 to 12 years, when school performance deteriorated and slowness, rigidity, and frequent falls developed. Examination revealed bradykinesia, subtle postural/action tremor, cogwheel rigidity, spasticity, upward gaze palsy, smooth pursuit with saccadic intrusions, and dementia. Additional signs included facial-faucial-finger minimyoclonus, absent postural reflexes, visual/auditory hallucinations, and insomnia. Levodopa response could not be fully judged in this family. T2* magnetic resonance imaging sequences revealed marked diffuse hypointensity of the caudate (head and body) and lenticular nucleus bilaterally. Disease progression was slow including epilepsy, cachexia, and anarthria. Four affected members died after 28.5 ± 5.5 (mean ± SD) years of disease. Two heterozygous carriers, the mother and eldest sibling, showed jerky perioral muscle contractions and clumsiness of hand movements. There was no significant correlation between DNA methylation of the ATP13A2 promoter region and disease progression. The marked caudate and lenticular nucleus T2*-hypointensity suggests that KRS might belong to the family of neurodegenerative diseases associated with brain iron accumulation. PMID:20683840

  13. Potential therapeutic target for malignant paragangliomas: ATP synthase on the surface of paraganglioma cells

    Science.gov (United States)

    Fliedner, Stephanie MJ; Yang, Chunzhang; Thompson, Eli; Abu-Asab, Mones; Hsu, Chang-Mei; Lampert, Gary; Eiden, Lee; Tischler, Arthur S; Wesley, Robert; Zhuang, Zhengping; Lehnert, Hendrik; Pacak, Karel

    2015-01-01

    F1FoATP synthase (ATP synthase) is a ubiquitous enzyme complex in eukaryotes. In general it is localized to the mitochondrial inner membrane and serves as the last step in the mitochondrial oxidative phosphorylation of ADP to ATP, utilizing a proton gradient across the inner mitochondrial membrane built by the complexes of the electron transfer chain. However some cell types, including tumors, carry ATP synthase on the cell surface. It was suggested that cell surface ATP synthase helps tumor cells thriving on glycolysis to survive their high acid generation. Angiostatin, aurovertin, resveratrol, and antibodies against the α and β subunits of ATP synthase were shown to bind and selectively inhibit cell surface ATP synthase, promoting tumor cell death. Here we show that ATP synthase β (ATP5B) is present on the cell surface of mouse pheochromocytoma cells as well as tumor cells of human SDHB-derived paragangliomas (PGLs), while being virtually absent on chromaffin primary cells from bovine adrenal medulla by confocal microscopy. The cell surface location of ATP5B was verified in the tissue of an SDHB-derived PGL by immunoelectron microscopy. Treatment of mouse pheochromocytoma cells with resveratrol as well as ATP5B antibody led to statistically significant proliferation inhibition. Our data suggest that PGLs carry ATP synthase on their surface that promotes cell survival or proliferation. Thus, cell surface ATP synthase may present a novel therapeutic target in treating metastatic or inoperable PGLs. PMID:26101719

  14. Retinoic acid isomers up-regulate ATP binding cassette A1 and G1 and cholesterol efflux in rat astrocytes: implications for their therapeutic and teratogenic effects.

    Science.gov (United States)

    Chen, Jing; Costa, Lucio G; Guizzetti, Marina

    2011-09-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (± 0.25), 3.6- (± 0.42), 4.1- (± 0.5), and 1.75- (± 0.43) fold, respectively, and Abcg1 by 2.1- (± 0.26), 2.2- (± 0.33), 2.5- (± 0.23), and 2.2- (± 0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions. PMID:21628419

  15. Synthesis and characterization in monkey of [{sup 11}C]SP203 as a radioligand for imaging brain metabotropic glutamate 5 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Simeon, Fabrice G.; Liow, Jeih-San; Zhang, Yi; Hong, Jinsoo; Gladding, Robert L.; Zoghbi, Sami S.; Innis, Robert B.; Pike, Victor W. [National Institutes of Health, Molecular Imaging Branch, National Institute of Mental Health, Bethesda, MD (United States)

    2012-12-15

    [{sup 18}F]SP203 (3-fluoro-5-(2-(2-([{sup 18}F]fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile) is an effective high-affinity and selective radioligand for imaging metabotropic 5 receptors (mGluR5) in human brain with PET. To provide a radioligand that may be used for more than one scanning session in the same subject in a single day, we set out to label SP203 with shorter-lived {sup 11}C (t{sub 1/2} = 20.4 min) and to characterize its behavior as a radioligand with PET in the monkey. Iodo and bromo precursors were obtained by cross-coupling 2-fluoromethyl-4-((trimethylsilyl)ethynyl)-1,3-thiazole with 3,5-diiodofluorobenzene and 3,5-dibromofluorobenzene, respectively. Treatment of either precursor with [{sup 11}C]cyanide ion rapidly gave [{sup 11}C]SP203, which was purified with high-performance liquid chromatography. PET was used to measure the uptake of radioactivity in brain regions after injecting [{sup 11}C]SP203 intravenously into rhesus monkeys at baseline and under conditions in which mGluR5 were blocked with 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP). The emergence of radiometabolites in monkey blood in vitro and in vivo was assessed with radio-HPLC. The stability of [{sup 11}C]SP203 in human blood in vitro was also measured. The iodo precursor gave [{sup 11}C]SP203 in higher radiochemical yield (>98 %) than the bromo precursor (20-52 %). After intravenous administration of [{sup 11}C]SP203 into three rhesus monkeys, radioactivity peaked early in brain (average 12.5 min) with a regional distribution in rank order of expected mGluR5 density. Peak uptake was followed by a steady decline. No radioactivity accumulated in the skull. In monkeys pretreated with MTEP before [{sup 11}C]SP203 administration, radioactivity uptake in brain was again high but then declined more rapidly than in the baseline scan to a common low level. [{sup 11}C]SP203 was unstable in monkey blood in vitro and in vivo, and gave predominantly less lipophilic radiometabolites

  16. Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days.

    Directory of Open Access Journals (Sweden)

    Daniela Santucci

    Full Text Available Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF and brain derived neurotrophic factor (BDNF, were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.

  17. Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

    International Nuclear Information System (INIS)

    The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EOR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the γ-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from 17O regiospecifically incorporated into the γ-phosphate group of ATP. By contrast, 17O in the α-phosphate or in the β-phosphate groups of ATP does not influence the spectrum. Experiments in 17O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme-Mn(II)-oxalate-Mg(II)-ATP complex suggests a scheme for the normal reverse reaction of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the γ-phosphate of ATP

  18. Brain Basics

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  20. Brain Basics

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    Full Text Available ... Neurons & Neural Circuits Neurons are the basic working unit of the brain and nervous system. These cells ... A nerve cell that is the basic, working unit of the brain and nervous system, which processes ...

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    Full Text Available ... Trials — Participants Statistics Help for Mental Illnesses Outreach Research Priorities Funding Labs at NIMH News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The ...

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  15. Brain Diseases

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    The brain is the control center of the body. It controls thoughts, memory, speech, and movement. It regulates the function of many organs. When the brain is healthy, it works quickly and automatically. However, ...

  16. Brain Basics

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  10. Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria - An amazing defence tool against hyperosmotic stress

    Directory of Open Access Journals (Sweden)

    Daniela eTrono

    2015-12-01

    Full Text Available In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about fifteen years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP. Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous and tissues/organs (etiolated and green have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane towards the matrix, so collapsing membrane potential (ΔΨ, the main component of the protonmotive force (Δp in plant mitochondria; moreover, cooperation between PmitoKATP and the K+/H+ antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate

  11. A Promising PET Tracer for Imaging of α7 Nicotinic Acetylcholine Receptors in the Brain: Design, Synthesis, and in Vivo Evaluation of a Dibenzothiophene-Based Radioligand

    Directory of Open Access Journals (Sweden)

    Rodrigo Teodoro

    2015-10-01

    Full Text Available Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET. We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10 has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

  12. Synthesis and characterization of 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) and structurally related compounds evidenced in seal blubber and brain.

    Science.gov (United States)

    von der Recke, Roland; Vetter, Walter

    2007-03-01

    The unknown compound UBC-1 previously described as the major organobromine contamination in the blubber extract of a hooded seal (Cystophora cristata) from the Barents Sea was identified as 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE). DPTE, which is the main component of the brominated flame retardant (BFR) Bromkal 73-5 PE, was synthesized by electrophilic addition of bromine to allyl-2,4,6-tribromophenyl ether (ATE). The chirality of DPTE was proven by gas chromatographic enantioseparation of the synthesized racemate. On the basis of GC/ECNI-MS ion chromatograms (m/z79 and 81), DPTE was the dominating organobromine compound in blubber and brain samples of hooded seals and harp seals (Phoca groenlandica) from the Barents and Greenland Seas. The concentrations of DPTE in blubber and brain were up to 470 and 340 microg/kg wet weight. Next to DPTE, the natural dibromo-trichloromonoterpene (MHC-1), the anthropogenic BDE 47 and BDE 99, as well as ATE, 3,5-dibromo-2-(2',4'-dibromo)-phenoxyanisole (6-MeO-BDE 47), 2-bromoallyl-2,4,6-tribromophenyl ether (BATE), and 4,6-dibromo-2-(2',4'-dibromo)-phenoxyanisole (2'-MeO-BDE 68) were present with decreasing relevance. BATE, which was detected for the first time in environmental samples, was synthesized from DPTE by E2 elimination. In brain samples of the harp seals, DPTE, ATE, and BATE were the most abundant organobromine compounds, whereas polybrominated diphenyl ethers (PBDEs) and MHC-1 were virtually absent. This indicated that DPTE, ATE, and BATE were able to penetrate the blood-brain barrier. The general co-occurrence of ATE and BATE in samples contaminated with DPTE support the hypothesis that these compounds are biotransformation products of DPTE. Anaerobic transformation studies of DPTE with super-reduced corrinoids resulted in the formation of ATE. Furthermore, 2,4,6-tribromophenol (TBP) and two other unknown minor transformation products were detected. PMID:17396646

  13. Brain Basics

    Medline Plus

    Full Text Available ... pituitary-adrenal (HPA) axis. Brain Basics in Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah ... having trouble coping with the stresses in her life. She began to think of suicide because she ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle-aged woman ... new memories. hypothalmic-pituitary-adrenal (HPA) axis —A brain-body ... stress. impulse —An electrical communication signal sent between neurons ...

  15. Brain Aneurysm

    Science.gov (United States)

    A brain aneurysm is an abnormal bulge or "ballooning" in the wall of an artery in the brain. They are sometimes called berry aneurysms because they ... often the size of a small berry. Most brain aneurysms produce no symptoms until they become large, ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... other cells guide neurons in forming various brain structures. Neighboring neurons make connections with each other and with distant nerve cells (via axons) to form brain circuits. These circuits control specific body functions such as sleep and speech. The brain continues ...

  17. Urinary ATP may be a dynamic biomarker of detrusor overactivity in women with overactive bladder syndrome

    OpenAIRE

    Miguel Silva-Ramos; Isabel Silva; Olga Oliveira; Sónia Ferreira; Maria Júlia Reis; José Carlos de Oliveira; Paulo Correia-de-Sá

    2013-01-01

    Background Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB) release more ATP than controls. Here, we compared the urinary ATP concent...

  18. Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F-1-ATP synthase

    OpenAIRE

    Böckmann, R.; Grubmueller, H.

    2002-01-01

    The mitochondrial membrane protein FoF1-ATP synthase synthesizes adenosine triphosphate (ATP), the universal currency of energy in the cell. This process involves mechanochemical energy transfer from rotating asymmetric gamma- 'stalk' to the three active sites of the F-1 unit, which drives the bound ATP out of the binding pocket. Here, the primary structural changes associated with this energy transfer in F-1- ATP synthase were studied with multi-nanosecond molecular dynamics simulations. By ...

  19. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

    OpenAIRE

    Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in...

  20. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    OpenAIRE

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support cha...