The variable colours of the fiddler crab Uca vomeris and their relation to background and predation.

Science.gov (United States)

Hemmi, Jan M; Marshall, Justin; Pix, Waltraud; Vorobyev, Misha; Zeil, Jochen

2006-10-01

Colour changes in fiddler crabs have long been noted, but a functional interpretation is still lacking. Here we report that neighbouring populations of Uca vomeris in Australia exhibit different degrees of carapace colours, which range from dull mottled to brilliant blue and white. We determined the spectral characteristics of the mud substratum and of the carapace colours of U. vomeris and found that the mottled colours of crabs are cryptic against this background, while display colours provide strong colour contrast for both birds and crabs, but luminance contrast only for a crab visual system. We tested whether crab populations may become cryptic under the influence of bird predation by counting birds overflying or feeding on differently coloured colonies. Colonies with cryptically coloured crabs indeed experience a much higher level of bird presence, compared to colourful colonies. We show in addition that colourful crab individuals subjected to dummy bird predation do change their body colouration over a matter of days. The crabs thus appear to modify their social signalling system depending on their assessment of predation risk.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

International Nuclear Information System (INIS)

Penefsky, H.S.

1985-01-01

Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

NARCIS (Netherlands)

Boyer, L.A.; Logie, C.; Bonte, E; Becker, P.B.; Wade, P.A.; Wolff, A.P.; Wu, C.; Imbalzano, A.N.; Peterson, C.L.

2000-01-01

ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates.

Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes.

NARCIS (Netherlands)

Horn, P.J.; Carruthers, L.M.; Logie, C.; Hill, D.A.; Solomon, M.J.; Wade, P.A.; Imbalzano, A.N.; Hansen, J.; Peterson, C.L.

2002-01-01

Members of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of

Evidence that two ATP-dependent (Lon proteases in Borrelia burgdorferi serve different functions.

Directory of Open Access Journals (Sweden)

James L Coleman

2009-11-01

Full Text Available The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH(2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host.

ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

Directory of Open Access Journals (Sweden)

Alexandra Lusser

2011-10-01

Full Text Available ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

Connexin hemichannel-mediated CO2-dependent release of ATP in the medulla oblongata contributes to central respiratory chemosensitivity

Science.gov (United States)

Huckstepp, Robert T R; id Bihi, Rachid; Eason, Robert; Spyer, K Michael; Dicke, Nikolai; Willecke, Klaus; Marina, Nephtali; Gourine, Alexander V; Dale, Nicholas

2010-01-01

Arterial , a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of in the blood and brain, yet the mechanisms by which the brain senses CO2 remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO2-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO2-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of . ATP release is independent of extracellular Ca2+ and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO2-dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO2-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO2-dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in is an important mechanism contributing to central respiratory chemosensitivity. PMID:20736421

Proteus mirabilis uroepithelial cell adhesin (UCA) fimbria plays a role in the colonization of the urinary tract.

Science.gov (United States)

Pellegrino, Rafael; Scavone, Paola; Umpiérrez, Ana; Maskell, Duncan J; Zunino, Pablo

2013-03-01

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Proteus mirabilis is an opportunistic pathogen, capable of causing severe UTIs, with serious kidney damage that may even lead to death. Several virulence factors are involved in the pathogenicity of this bacterium. Among these, adherence to the uroepithelium mediated by fimbriae appears to be a significant bacterial attribute related to urovirulence. Proteus mirabilis expresses several types of fimbriae that could be involved in the pathogenesis of UTI, including uroepithelial cell adhesin (UCA). In this report, we used an uropathogenic P. mirabilis wild-type strain and an isogenic ucaA mutant unable to express UCA to study the pathogenic role of this fimbria in UTI. Ability of the mutant to adhere to desquamated uroepithelial cells and to infect mice using different experimental UTI models was significantly impaired. These results allow us to conclude that P. mirabilis UCA plays an important role in the colonization of the urinary tract. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Gut-Associated Microbial Symbionts of the Marsh Fiddler Crab, Uca Pugnax

National Research Council Canada - National Science Library

Gunman, Lara K

2004-01-01

.... The overarching goal of this thesis was to characterize the ecology and genetic diversity of resident gut microbes to advance our understanding of their interactions with their host, the marsh fiddler crab, Uca pugnax...

ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro

International Nuclear Information System (INIS)

Woehle, D.L.; Lueddecke, B.A.; Ludden, P.W.

1990-01-01

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [α- 32 P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [α- 32 P]ATP- and [α- 32 P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [α- 32 P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes. A 32 P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H 3 32 PO 4 -grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification

Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

International Nuclear Information System (INIS)

Yang, S.D.; Fong, Y.L.

1985-01-01

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32 P-labeled myelin basic protein (MBP) and [ 32 P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

Science.gov (United States)

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

Science.gov (United States)

Bele, Tanja; Fabbretti, Elsa

2016-08-01

P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

Effects of acidified seawater on coral calcification and variations of U/Ca ratio in their skeletons

Science.gov (United States)

Inoue, M.; Ozaki, S.; Iguchi, A.; Sakai, K.; Suzuki, A.; Kawahata, H.

2011-12-01

The rising CO2 concentration in the atmosphere is changing the carbonate chemistry of the ocean. Elevated partial pressure of CO2 (pCO2) has caused significant decrease in surface seawater pH and carbonate ion concentration. Therefore, ocean acidification has a negative effect on calcification of marine calcifying organisms. Especially, hermatypic corals are dominant organisms in coral reef ecosystems, so their calcificication is a key to determine the health of reef ecosystems. On the other hand, recent study has suggested that there is a negative correlation between U/Ca ratio in coral skeleton and seawater pH, based on the culture experiment using primary polyps of Acropora digitifera. In this study, primary polyps and adult colonies of A. digitifera and adult colonies of Porites australiensis, which are the dominant species around the Ryukyu Islands, Japan, were reared in seawater with different pCO2 (300, 400, 600, 800, 1000ppm) and pH (7.4, 7.6, 8.0) settings controlled by CO2 bubbling. Calcification rate of adult coral was estimated by buoyant method, while skeletal growth of polyps was evaluated by measuring the dry weight of each skeleton after the experiments. In order to evaluate the relationship between U/Ca ratios in coral skeletons and seawater pH, U/Ca ratios in reared corals were analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The results of A. digitifera showed that the growth rate of adult corals had no significant correlation against pCO2, but dry weight of polyp skeletons decreased with increase in pCO2. Growth rate of P. australiensis typically showed a positive correlation with pH. However, growth rates were different among colonies, suggesting that their responses to acidification may vary among the colonies. Regarding the variations of U/Ca ratios, there were positive correlations between U/Ca ratios in adults of A. digitifera and P. australiensis and seawater pCO2 (pH), while no relation was observed in polyp corals.

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

Science.gov (United States)

Park, Yun Yeon; Ahn, Ju-Hyun; Cho, Min-Guk; Lee, Jae-Ho

2018-04-27

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN 3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/C Cdc20 , we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/C Cdh1 , but not APC/C Cdc20 , facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/C Cdh1 -dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

International Nuclear Information System (INIS)

Fisher, G.J.; Kelley, L.K.; Smith, C.H.

1987-01-01

As a first step in understanding the cellular basis of maternal-fetal calcium transfer, the authors examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake. This uptake required magnesium, was not significantly affected by Na + or K + (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. They conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer

ATP-dependent human RISC assembly pathways.

Science.gov (United States)

Yoda, Mayuko; Kawamata, Tomoko; Paroo, Zain; Ye, Xuecheng; Iwasaki, Shintaro; Liu, Qinghua; Tomari, Yukihide

2010-01-01

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

ATP Release Channels

Directory of Open Access Journals (Sweden)

Akiyuki Taruno

2018-03-01

Full Text Available Adenosine triphosphate (ATP has been well established as an important extracellular ligand of autocrine signaling, intercellular communication, and neurotransmission with numerous physiological and pathophysiological roles. In addition to the classical exocytosis, non-vesicular mechanisms of cellular ATP release have been demonstrated in many cell types. Although large and negatively charged ATP molecules cannot diffuse across the lipid bilayer of the plasma membrane, conductive ATP release from the cytosol into the extracellular space is possible through ATP-permeable channels. Such channels must possess two minimum qualifications for ATP permeation: anion permeability and a large ion-conducting pore. Currently, five groups of channels are acknowledged as ATP-release channels: connexin hemichannels, pannexin 1, calcium homeostasis modulator 1 (CALHM1, volume-regulated anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR anion channels, and maxi-anion channels (MACs. Recently, major breakthroughs have been made in the field by molecular identification of CALHM1 as the action potential-dependent ATP-release channel in taste bud cells, LRRC8s as components of VRACs, and SLCO2A1 as a core subunit of MACs. Here, the function and physiological roles of these five groups of ATP-release channels are summarized, along with a discussion on the future implications of understanding these channels.

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

DEFF Research Database (Denmark)

Kowal, Justyna Magdalena

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......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 P2......, 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...

A key to the "X-Species" of North American fiddler crabs (genus uca)

NARCIS (Netherlands)

Hagen, von H.-O.

1980-01-01

Up to the late sixties of this century the number of species of the genus Uca occurring on the East and Gulf coasts of North America seemed rather well established. Usually ten species were listed: U. burgersi Holthuis ( = U. affinis (Streets)), U. leptodactyla Rathbun, U. minax (Le Conte), U.

A simple and versatile system for the ATP-dependent assembly of chromatin.

Science.gov (United States)

Khuong, Mai T; Fei, Jia; Cruz-Becerra, Grisel; Kadonaga, James T

2017-11-24

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

Science.gov (United States)

Xiong, Yingfei; Teng, Sasa; Zheng, Lianghong; Sun, Suhua; Li, Jie; Guo, Ning; Li, Mingli; Wang, Li; Zhu, Feipeng; Wang, Changhe; Rao, Zhiren; Zhou, Zhuan

2018-02-28

Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca 2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca 2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca 2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca 2+ independent single large non-quantal ATP release occurred, in contrast to the Ca 2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength

DEFF Research Database (Denmark)

Hansen, Emilie Louise; Sozer, Esin Bengisu; Romeo, Stefania

2015-01-01

death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. METHODS: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability...... was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. RESULTS: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p...-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field. GENERAL SIGNIFICANCE: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials....

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

Directory of Open Access Journals (Sweden)

Hayoz Sébastien

2012-05-01

Full Text Available 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 Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP

The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth

DEFF Research Database (Denmark)

Købmann, Brian Jensen; Westerhoff, H.V.; Snoep, J.L.

2002-01-01

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch...... cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis...... (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells....

Climatic Constraints on Growth Rate and Geochemistry (Sr/Ca and U/Ca) of the Coral Siderastrea stellata in the Southwest Equatorial Atlantic (Rocas Atoll, Brazil)

Science.gov (United States)

Evangelista, H.; Sifeddine, A.; Corrège, T.; Servain, J.; Dassié, E. P.; Logato, R.; Cordeiro, R. C.; Shen, C.-C.; Le Cornec, F.; Nogueira, J.; Segal, B.; Castagna, A.; Turcq, B.

2018-03-01

Although relatively rare compared to similar latitudes in the Pacific or Indian Oceans, massive coral colonies are present in the Tropical/Equatorial Southwestern Atlantic Ocean. However, detailed geochemical compositions of these corals are still largely unknown. In this work, we present growth rates, Sr/Ca, and U/Ca ratios of the coral colony (Siderastrea stellata) sampled at Rocas Atoll, off the Brazilian coast. These variables are primarily affected by sea surface temperature (SST) at seasonal scale, and by wind stress at interannual scale, these results represent a broad new finding. A lower significance at the interannual time scale between Sr/Ca and U/Ca with respect to SST is attributed to the low SST amplitude closed to Equator. An investigation on the dependence of coral growth rates with respect to the "cloud shading effect" promoted by the Intertropical Convergence Zone (ITCZ) does not show significant influence. Additionally, rain seems to act on local geochemistry of Sr/Ca ratios and growth rate at the decadal scale.

[Mg2+, ATP-dependent plasma membrane calcium pump of smooth muscle cells. I. Structural organization and properties].

Science.gov (United States)

Veklich, T O; Mazur, Iu Iu; Kosterin, S O

2015-01-01

Tight control of cytoplasm Ca2+ concentration is essential in cell functioning. Changing of Ca2+ concentration is thorough in smooth muscle cells, because it determines relaxation/constraint process. One of key proteins which control Ca2+ concentration in cytoplasm is Mg2+, ATP-dependent plasma membrane calcium pump. Thus, it is important to find compoumds which allowed one to change Mg2+, ATP-dependent plasma membrane calcium pump activity, as long as this topic is of current interest in biochemical research which regards energy and pharmacomechanical coupling mechanism of muscle excitation and contraction. In this article we generalized literatute and own data about properties of smooth muscle cell plasma membrane Ca(2+)-pump. Stuctural oganization, kinetical properties and molecular biology are considered.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

Science.gov (United States)

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

International Nuclear Information System (INIS)

Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

1986-01-01

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

ATP, IMP, and glycogen in cod muscle at onset and during development of rigor mortis depend on the sampling location

DEFF Research Database (Denmark)

Cappeln, Gertrud; Jessen, Flemming

2002-01-01

Variation in glycogen, ATP, and IMP contents within individual cod muscles were studied in ice stored fish during the progress of rigor mortis. Rigor index was determined before muscle samples for chemical analyzes were taken at 16 different positions on the fish. During development of rigor......, the contents of glycogen and ATP decreased differently in relation to rigor index depending on sampling location. Although fish were considered to be in strong rigor according to the rigor index method, parts of the muscle were not in rigor as high ATP concentrations were found in dorsal and tall muscle....

The effect of salinity on larval development of Uca tangeri (Eydoux, 1835 (Brachyura: Ocypodidae and new findings of the zoeal morphology

Directory of Open Access Journals (Sweden)

Eduardo D. Spivak

2009-06-01

Full Text Available In order to evaluate their tolerance to low salinities, zoeae of the fiddler crab Uca tangeri from the Rio San Pedro population (southwestern Spain were reared in the laboratory at 20ºC and at three salinities (16, 24 and 32. The zoeal development was completed at 24 and 32 but the crabs died as zoea I or zoea II, and very rarely as zoea III, at 16; tolerance to low salinities varied among clutches produced by different females. The duration of the first zoeal stage and of the complete zoeal development was shorter at 32. Our observations showed that the zoeae of U. tangeri could not tolerate retention in the mesohaline water of estuaries, and that export to oceanic waters would be optimal for their successful development. Survival at 24 suggests that larvae could also develop in polyhaline conditions if they were retained in the ocean-estuary interface. The presence of an additional zoeal stage (zoea VI was observed in some individuals and associated with unfavourable combinations of temperature and salinity. In addition, some previously omitted aspects of zoeal morphology were re-described and illustrated, providing new evidence which supports the basal position of this species in a proto-Atlantic origin of the genus Uca. It is proposed that the differences between Uca tangeri and the rest of the Uca species should be highlighted, and that it should be placed in its own genus as Afruca tangeri.

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. Further......, despite differences in overall architecture, both appear to operate by an alternating access mechanism and during transport they might allow access of phospholipids to the internal part of the transmembrane domain. The latter feature is obvious for ABC transporters, but phospholipids and other hydrophobic...

Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

Science.gov (United States)

Schühle, Karola

2012-01-01

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

ATP-independent DNA synthesis in Vaccinia-infected L cells

International Nuclear Information System (INIS)

Berger, N.A.; Kauff, R.A.; Sikorski, G.W.

1978-01-01

Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin

Science.gov (United States)

Polyoxin (POL) is an unusual nucleoside antibiotic, in which peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG muta...

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

Science.gov (United States)

de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

2011-02-01

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

A futile cycle, formed between two ATP-dependant γ-glutamyl cycle ...

Indian Academy of Sciences (India)

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 ...

ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

DEFF Research Database (Denmark)

Zhao, A.; Gray, F. C; MacNeill, S. A.

2006-01-01

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile....... volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that Lig...

A case of malformation on the third maxilliped of Uca rapax (Smith, 1870 (Decapoda: Ocypodidae

Directory of Open Access Journals (Sweden)

Carlos Lira

Full Text Available This paper evaluates the malformation in the left third maxilliped of a specimen of the fiddler crab Uca rapax from Venezuela. There are some hypotheses and the cause of the malformation remains unknown, but the results are indicative that is most likely due to errors in morphogenetic processes.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

Science.gov (United States)

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Modeling steady state SO2-dependent changes in capillary ATP concentration using novel O2 micro-delivery methods

Science.gov (United States)

Ghonaim, Nour W.; Fraser, Graham M.; Ellis, Christopher G.; Yang, Jun; Goldman, Daniel

2013-01-01

Adenosine triphosphate (ATP) is known to be released from the erythrocyte in an oxygen (O2) dependent manner. Since ATP is a potent vasodilator, it is proposed to be a key regulator in the pathway that mediates micro-vascular response to varying tissue O2 demand. We propose that ATP signaling mainly originates in the capillaries due to the relatively long erythrocyte transit times in the capillary and the short ATP diffusion distance to the electrically coupled endothelium. We have developed a computational model to investigate the effect of delivering or removing O2 to limited areas at the surface of a tissue with an idealized parallel capillary array on total ATP concentration. Simulations were conducted when exposing full surface to perturbations in tissue O2 tension (PO2) or locally using a circular micro-outlet (~100 μm in diameter), a square micro-slit (200 × 200 μm), or a rectangular micro-slit (1000 μm wide × 200 μm long). Results indicated the rectangular micro-slit has the optimal dimensions for altering hemoglobin saturations (SO2) in sufficient number capillaries to generate effective changes in total [ATP]. This suggests a threshold for the minimum number of capillaries that need to be stimulated in vivo by imposed tissue hypoxia to induce a conducted micro-vascular response. SO2 and corresponding [ATP] changes were also modeled in a terminal arteriole (9 μm in diameter) that replaces 4 surface capillaries in the idealized network geometry. Based on the results, the contribution of terminal arterioles to the net change in [ATP] in the micro-vascular network is minimal although they would participate as O2 sources thus influencing the O2 distribution. The modeling data presented here provide important insights into designing a novel micro-delivery device for studying micro-vascular O2 regulation in the capillaries in vivo. PMID:24069001

Formação de Professores que Ensinam Matemática no Contexto da Cibercultura: Estudo em uma Escola UCA

Directory of Open Access Journals (Sweden)

Dennys Leite Maia

2014-09-01

Full Text Available Nas últimas décadas, com o advento das tecnologias digitais, a sociedade tem passado por uma reconfiguração. As relações entre tais tecnologias e a cultura contemporânea é definida como Cibercultura. Nessa perspectiva, este trabalho tem como objetivo analisar de que forma as interações mediadas pelas tecnologias digitais contribuem para o desenvolvimento profissional do professor de Matemática, particularmente sobre o laptop educacional, do Projeto Um Computador por Aluno (UCA. Como método de pesquisa, utilizamos elementos da Pesquisa Colaborativa. A pesquisa foi realizada em uma escola de Ensino Fundamental, participante do Projeto UCA, localizada em Fortaleza-CE.

Estimation of PMI depends on the changes in ATP and its degradation products.

Science.gov (United States)

Mao, Shiwei; Fu, Gaowen; Seese, Ronald R; Wang, Zhen-Yuan

2013-09-01

Estimating the time since death, or postmortem interval (PMI), has been one of the biggest difficulties in modern forensic investigation. This study tests if the concentrations of breakdown products of adenosine triphosphate (ATP) correlate with PMI in multiple organs from rat. Brains, spleens, and kidneys of rats were harvested at different time points in carcasses maintained at 4°C or 20°C. High Performance Liquid Chromatography (HPLC) was used to quantify concentrations of metabolites related to ATP degradation. A K value (Kv=100×(Hx+HxR)/(ATP+ADP+AMP+IMP+HxR+Hx)) was calculated and correlated with PMI for each organ and temperature. The results indicate that the K value is a robust index for the estimation of PMI based on highly significant linear correlations between PMI and concentrations of ATP breakdown products. Compared with other current research methods, the changing tendency of ATP and its degradation products may be potentially a better way for the estimation of PMI in medico-legal practice. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks

Directory of Open Access Journals (Sweden)

Daniel eGoldman

2012-07-01

Full Text Available Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100ms that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2. The model further predicts how insulin, at concentrations found in prediabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Limb regeneration and molting processes under chronic methoprene exposure in the mud fiddler crab, Uca pugnax.

Science.gov (United States)

Stueckle, Todd A; Likens, Jason; Foran, Christy M

2008-04-01

Insect growth regulator application for wetland mosquito control remains controversial due to the potential for disruption of normal development and growth processes in non-target crustaceans and beneficial arthropods, e.g. Apis mellifera. Concerns include slow-release methoprene formulations and its environmental breakdown products which mimic an endogenous crustacean hormone and retinoids, respectively. Our primary objective was to evaluate the effect that a chronic methoprene exposure would have on male and female Uca pugnax limb regeneration and molting. After single limb autonomy, limb growth and molt stage were monitored every two days while eyestalk ablation was used to induce proecdysis. Dorsal carapace was collected 6 days post-molt to determine protein and chitin content. In post-molt crabs, methoprene-exposed individuals displayed lower percent gain in body weight. Male crabs lost more weight per body volume than females, took significantly longer to proceed through proecdysis than females exposed to 0.1 microg/L methoprene and exhibited significantly elevated frequency for abnormal limb formation at 1.0 microg/L while females displayed no such trend. Methoprene did not significantly alter extractable exoskeleton protein or chitin content. However, variable water-soluble protein expression increased with exposure at 1.0 microg/L (1 ppb) which contributed to overall variability in total protein content. Our findings suggest that adult male U. pugnax possess greater sensitivity to chronic methoprene exposure during limb regeneration and molting, potentially affecting their post-molt fitness. Furthermore, methoprene has the potential to impact post-molt biomass and exocuticle quality.

Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans.

Science.gov (United States)

Gay, Sean C; Fribourgh, Jennifer L; Donohoue, Paul D; Segel, Irwin H; Fisher, Andrew J

2009-09-01

The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria.

Science.gov (United States)

Nocek, Boguslaw; Kochinyan, Samvel; Proudfoot, Michael; Brown, Greg; Evdokimova, Elena; Osipiuk, Jerzy; Edwards, Aled M; Savchenko, Alexei; Joachimiak, Andrzej; Yakunin, Alexander F

2008-11-18

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

Age-dependent changes of cerebral copper metabolism in Atp7b -/- knockout mouse model of Wilson's disease by [64Cu]CuCl2-PET/CT.

Science.gov (United States)

Xie, Fang; Xi, Yin; Pascual, Juan M; Muzik, Otto; Peng, Fangyu

2017-06-01

Copper is a nutritional metal required for brain development and function. Wilson's disease (WD), or hepatolenticular degeneration, is an inherited human copper metabolism disorder caused by a mutation of the ATP7B gene. Many WD patients present with variable neurological and psychiatric symptoms, which may be related to neurodegeneration secondary to copper metabolism imbalance. The objective of this study was to explore the feasibility and use of copper-64 chloride ([ 64 C]CuCl 2 ) as a tracer for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD using an Atp7b -/- knockout mouse model of WD and positron emission tomography/computed tomography (PET/CT) imaging. Continuing from our recent study of biodistribution and radiation dosimetry of [ 64 C]CuCl 2 in Atp7b -/- knockout mice, PET quantitative analysis revealed low 64 Cu radioactivity in the brains of Atp7b -/- knockout mice at 7th weeks of age, compared with 64 Cu radioactivity in the brains of age- and gender-matched wild type C57BL/6 mice, at 24 h (h) post intravenous injection of [ 64 C]CuCl 2 as a tracer. Furthermore, age-dependent increase of 64 Cu radioactivity was detected in the brains of Atp7b -/- knockout mice from the 13th to 21th weeks of age, based on the data derived from a longitudinal [ 64 C]CuCl 2 -PET/CT study of Atp7b -/- knockout mice with orally administered [ 64 Cu]CuCl 2 as a tracer. The findings of this study support clinical use of [ 64 Cu]CuCl 2 -PET/CT imaging as a tool for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD patients presenting with variable neurological and psychiatric symptoms.

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)

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

1987-01-01

Isolated rat pancreatic islets were pulse-labeled for 5 min with [ 3 H]leucine then chased for 25 min, during which time endogenously labeled [ 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 [ 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

X-ray effects on the activity of a Mg2+-dependent, Na+- and K+-activable microsomal membrane ATP-ase system

International Nuclear Information System (INIS)

Froehlich, D.

1978-01-01

The bahviour of a Mg 2+ -dependent, Na + - and K + -activable ATP-ase sytem on irradiation was investigated using a microsome fraction of guinea pig myocardial cells prepared by fractionated centrifugation. The Na + - and K + -activable component, transport-ATPase, was particularly radiation-sensitive. Three stages of development were observed for a 1,500 R radiation damage until 24 h p.r.. In the first stage, until 30 minutes p.r., the activity of transport-ATP-ase was inhibited. This was followed by repair processes which had reached a peak value clearly higher than the control values at 4 hours p.r.. In the third stage, the activity was reduced again; 15 and 24 hours after termination of exposure, values again were nearly the same as after 30 minutes where a maximum was observed for this radiation dose. Radiation-induced electrolyte displacements, active transport, and radiation-induced inhibition of transport-ATP-ase were correlated and discussed; the assumption was that changes in, the electrolyte conditions in the membranes on irradiation are at least partly due to the described inhibition of transport-ATP-ase. (orig./AJ) [de

Robotic crabs reveal that female fiddler crabs are sensitive to changes in male display rate.

Science.gov (United States)

Mowles, Sophie L; Jennions, Michael D; Backwell, Patricia R Y

2018-01-01

Males often produce dynamic, repetitive courtship displays that can be demanding to perform and might advertise male quality to females. A key feature of demanding displays is that they can change in intensity: escalating as a male increases his signalling effort, but de-escalating as a signaller becomes fatigued. Here, we investigated whether female fiddler crabs, Uca mjoebergi , are sensitive to changes in male courtship wave rate. We performed playback experiments using robotic male crabs that had the same mean wave rate, but either escalated, de-escalated or remained constant. Females demonstrated a strong preference for escalating robots, but showed mixed responses to robots that de-escalated ('fast' to 'slow') compared to those that waved at a constant 'medium' rate. These findings demonstrate that females can discern changes in male display rate, and prefer males that escalate, but that females are also sensitive to past display rates indicative of prior vigour. © 2018 The Authors.

The Martyrs of the UCA: demand and grace. Social commitment of the Catholic University

Directory of Open Access Journals (Sweden)

Jon Sobrino, SJ

2010-12-01

Full Text Available Hace veinte años asesinaron a mis hermanos jesuitas de la UCA, a Julia Elba y a Celina. Yo me encontraba en Tailandia, y a mi regreso a El Salvador tenía que pasar por San Francisco. En el aeropuerto me esperaban –con rostros impávidos- Steve Prevett y Peggy O’Grady. En las calles de San Francisco, con un parlante en la mano, Paul Locatelli condenaba los asesinatos, Tessa Rouverol le acompañaba.

Effect of hydrogen peroxide on the main kinetic parameters of ATP hydrolysis by ouabain sensitive Na+, K+-ATP-ase in spermatozoa of infertile men

Directory of Open Access Journals (Sweden)

Р. В. Фафула

2017-12-01

Full Text Available Background: It is known that Na+,K+-ATP-ase plays important role in physiology of spermatozoa including their motility. Na+,K+-ATP-ase is one of the targets for reactive oxygen species. Hyperproduction of reactive oxygen species can damage sperm cells and it is considered to be as one of the mechanisms of male infertility. Objectives: To evaluate the H2O2 effect on the main kinetic parameters of ATP hydrolysis by ouabain-sensitive Na+,K+-ATPase of spermatozoa of fertile (normozoospermia and infertility men (asthenozoospermia. Materials and methods: Na+, K+-ATP-ase activity was determined spectrophotometrically by production of Pi. Concentration dependencies ware linearized in Lineweaver-Burk plot. Results: Effective inhibitory effect of H2O2 on ouabain-sensitive Na+,K+-ATP-ase activity of sperm cells of fertile and infertile men was demonstrated. The effects of H2O2 on the main kinetic parameters of the ATP hydrolysis with the involvement of Na+, K+-ATP-ase was studied. In the whole range of studied concentrations of ATP the Na+, K+-ATP-ase activity of spermatozoa of fertile and infertile men was reduced in the presence of H2O2 in the incubation medium. However, the optimal activity of the Na+, K+-ATP-ase activity of sperm cells in both normozoospermic and asthenozoospermic men was observed in the presence of 5 mM ATP in the incubation medium. By linearization of concentration curves in Lineweaver-Burk plot the main kinetic parameters of Na+, K+-activated, Mg2+-dependent ATP hydrolysis in the sperm cells of fertile and infertile men were determined. Under the effect of H2O2, the affinity constant of enzyme to ATP in normozoospermic and asthenozoospermic men increases several times. The initial maximum rate of ATP hydrolysis was significantly reduced only in the spermatozoa of fertile men with normozoospermia. Conclusions: Under conditions of H2O2-induced oxidative stress the inhibition of ouabain-sensitive Na+,K+-ATP-ase activity in sperm cells

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

Science.gov (United States)

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

2013-10-08

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

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

Science.gov (United States)

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

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

Population structure and fecundity in Uca virens Salmon & Atsaides, 1968 (Decapoda: Ocypodidae in southern Tamiahua Lagoon, Veracruz, Mexico

Directory of Open Access Journals (Sweden)

Víctor del Castillo

2015-03-01

Full Text Available Fiddler crabs are common decapods of intertidal zones; thirteen species have been reported along the coast of the Gulf of Mexico, inhabiting different zones. For the southern region of the Tamiahua Lagoon, Veracruz, Mexico, five species have been identified: Uca panacea, U. rapax, U. spinicarpa, U. virens and U. vocator particularly in the southern region of the Tamiahua Lagoon, Veracruz, Mexico. Here we analysed the fecundity of U. virens in 25 ovigerous females out of 387 individuals collected between December 2008 and December 2009 during eight field trips and from five collection sites. Morphological measurements like carapace length (CL and carapace width (CW, and total wet weight (TWW for all individuals collected were taken, and total egg number was counted for ovigerous females . The total population was divided in nine size class intervals. Total egg number varied between 3,617 and 41,099. Egg number increased with CW (ranging from 9.0 to 18.6 mm CW and TWW. Values of fecundity observed for U. virens in Tamiahua Lagoon vs other Uca species were similar to results reported in literature.

Dynamics of shear-induced ATP release from red blood cells.

Science.gov (United States)

Wan, Jiandi; Ristenpart, William D; Stone, Howard A

2008-10-28

Adenosine triphosphate (ATP) is a regulatory molecule for many cell functions, both for intracellular and, perhaps less well known, extracellular functions. An important example of the latter involves red blood cells (RBCs), which help regulate blood pressure by releasing ATP as a vasodilatory signaling molecule in response to the increased shear stress inside arterial constrictions. Although shear-induced ATP release has been observed widely and is believed to be triggered by deformation of the cell membrane, the underlying mechanosensing mechanism inside RBCs is still controversial. Here, we use an in vitro microfluidic approach to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolution. We demonstrate that there is a sizable delay time between the onset of increased shear stress and the release of ATP. This response time decreases with shear stress, but surprisingly does not depend significantly on membrane rigidity. Furthermore, we show that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is commensurate with a characteristic membrane relaxation time determined from observations of the cell deformation by using high-speed video. Taken together our results suggest a model wherein the retraction of the spectrin-actin cytoskeleton network triggers the mechanosensitive ATP release and a shear-dependent membrane viscosity controls the rate of release.

Visual ergonomic aspects of glare on computer displays: glossy screens and angular dependence

Science.gov (United States)

Brunnström, Kjell; Andrén, Börje; Konstantinides, Zacharias; Nordström, Lukas

2007-02-01

Recently flat panel computer displays and notebook computer are designed with a so called glare panel i.e. highly glossy screens, have emerged on the market. The shiny look of the display appeals to the costumers, also there are arguments that the contrast, colour saturation etc improves by using a glare panel. LCD displays suffer often from angular dependent picture quality. This has been even more pronounced by the introduction of Prism Light Guide plates into displays for notebook computers. The TCO label is the leading labelling system for computer displays. Currently about 50% of all computer displays on the market are certified according to the TCO requirements. The requirements are periodically updated to keep up with the technical development and the latest research in e.g. visual ergonomics. The gloss level of the screen and the angular dependence has recently been investigated by conducting user studies. A study of the effect of highly glossy screens compared to matt screens has been performed. The results show a slight advantage for the glossy screen when no disturbing reflexes are present, however the difference was not statistically significant. When disturbing reflexes are present the advantage is changed into a larger disadvantage and this difference is statistically significant. Another study of angular dependence has also been performed. The results indicates a linear relationship between the picture quality and the centre luminance of the screen.

De Novo Mutations in CHD4, an ATP-Dependent Chromatin Remodeler Gene, Cause an Intellectual Disability Syndrome with Distinctive Dysmorphisms

NARCIS (Netherlands)

Weiss, Karin; Terhal, Paulien A; Cohen, Lior; Bruccoleri, Michael; Irving, Melita; Martinez, Ariel F; Rosenfeld, Jill A; Machol, Keren; Yang, Yaping; Liu, Pengfei; Walkiewicz, Magdalena; Beuten, Joke; Gomez-Ospina, Natalia; Haude, Katrina; Fong, Chin-To; Enns, Gregory M; Bernstein, Jonathan A; Fan, Judith; Gotway, Garrett; Ghorbani, Mohammad; van Gassen, Koen; Monroe, Glen R; van Haaften, Gijs; Basel-Vanagaite, Lina; Yang, Xiang-Jiao; Campeau, Philippe M; Muenke, Maximilian

2016-01-01

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2β, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we

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

Science.gov (United States)

Dudev, Todor; Grauffel, Cédric; Lim, Carmay

2017-02-01

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

The Role of ATP in Sleep Regulation

Directory of Open Access Journals (Sweden)

Sachiko eChikahisa

2011-12-01

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

Multiple aspects of ATP-dependent nucleosome translocation by RSC and Mi-2 are directed by the underlying DNA sequence.

Directory of Open Access Journals (Sweden)

Joke J F A van Vugt

Full Text Available BACKGROUND: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. METHODOLOGY/PRINCIPAL FINDINGS: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type and native RSC from yeast (SWI/SNF-type repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. CONCLUSIONS/SIGNIFICANCE: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase

Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

International Nuclear Information System (INIS)

Mithieux, G.; Rousset, B.

1989-01-01

We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

Science.gov (United States)

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Tulika Munshi

Full Text Available ATP-dependent Mur ligases (Mur synthetases play essential roles in the biosynthesis of cell wall peptidoglycan (PG as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Nisin depletes ATP and proton motive force in mycobacteria.

Science.gov (United States)

Chung, H J; Montville, T J; Chikindas, M L

2000-12-01

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

Science.gov (United States)

Francis, Brian R; White, Karen H; Thorsness, Peter E

2007-04-01

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

Synergistic binding of glucose and aluminium ATP to hexokinase from Saccharomyces cerevisiae.

Science.gov (United States)

Woolfitt, A R; Kellett, G L; Hoggett, J G

1988-08-10

The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence. No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM. In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of [L]0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM. The presence of saturating concentrations of AlATP diminished [L]0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer. The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP. The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction.

Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

Science.gov (United States)

Durand, Maxim; Dubois, Florian; Dejou, Cécile; Durand, Eugénie; Danger, Richard; Chesneau, Mélanie; Brosseau, Carole; Guerif, Pierrick; Soulillou, Jean-Paul; Degauque, Nicolas; Eliaou, Jean-François; Giral, Magali; Bonnefoy, Nathalie; Brouard, Sophie

2018-05-01

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Inhibition of the cardiac ATP-dependent potassium current by KB-R7943.

Science.gov (United States)

Abramochkin, Denis V; Vornanen, Matti

2014-09-01

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium-calcium exchanger (NCX) with potential experimental and therapeutic use. However, in cardiomyocytes KB-R7943 also effectively blocks several K(+) currents including the delayed rectifier, IKr, and background inward rectifier, IK1. In the present study we analyze the effects of KB-R7943 on the ATP-dependent potassium current (IKATP) recorded by whole-cell patch-clamp in ventricular cardiomyocytes from a mammal (mouse) and a fish (crucian carp). IKATP was induced by external application of a mitochondrial uncoupler CCCP (3×10(-7) M) and internal perfusion of the cell with ATP-free pipette solution. A weakly inwardly rectifying current with a large outward component, recorded in the presence of CCCP, was blocked with 10(-5) M glibenclamide by 56.1±4.6% and 56.9±3.6% in crucian carp and mouse ventricular myocytes, respectively. In fish cardiomyocytes IKATP was blocked by KB-R7943 with an IC50 value of 3.14×10(-7) M, while in mammalian cells IC50 was 2.8×10(-6) M (PKB-R7943 inhibited CCCP-induced IKATP by 99.9±0.13% and 97.5±1.2% in crucian carp and mouse ventricular myocytes, respectively. In crucian carp the IKATP is about an order of magnitude more sensitive to KB-R7943 than the background IK1, but in mammals IKATP and IK1 are almost equally sensitive to KB-R7943. Therefore, the ability of KB-R7943 to block IKATP should be taken into account together with INCX inhibition when investigating possible cardioprotective effects of this compound. Copyright © 2014 Elsevier Inc. All rights reserved.

InP/ZnS Nanocrystals as Fluorescent Probes for the Detection of ATP

OpenAIRE

Massadeh, Salam; Nann, Thomas

2014-01-01

This article reports on a study on fluorescence adenosine triphosphate (ATP) detection by InP/ZnS quantum dots (QDs). We present a spectroscopic analysis displaying the effect of enzymatic reactions of glucose oxidase (GOX) and hexokinase (HEX) on the InP/ZnS quantum dots at physiological pH. The InP/ZnS quantum dots act as glucose sensors in the presence of GOX, Glu and ATP, and their luminescence quenches during the release of hydrogen peroxide from the reaction. However, in the presence of...

Levels of adenine nucleotides (ATP, ADP, AMP) and of inorganic phosphate in needles of Picea abies, representing different stages of development and of pollution dependence

Energy Technology Data Exchange (ETDEWEB)

Benz, T; Hampp, R; Horsch, F; Filby, G; Fund, N; Gross, S; Hanisch, B; Kilz, E; Seidel, A [comps.

1986-04-01

Levels of adenine nucleotides (ATP, ADP, AMP) and of inorganic phosphate in needles of Picea abies, representing different stages of development and of pollution dependence. Lyophilized needles of Picea abies (Kaelbelescheuer, southern Black Forest) were analyzed for their content of adenine nucleotides (ATP, ADP, AMP: AdN) and of inorganic phosphate (Psub(i)). The metabolite levels were related to needle age, vegetation period and degree of damage (chlorophyll content). The results were as follows: 1) With increasing needle age there is a general decrease in the total AdN-pool. This decrease is most pronounced in very young needles and occurs in both healthy and damaged tissue. 2) The ATP/ADP-ratio of damaged needle is significantly higher than that of healthy ones. 3) Both phosphorylation potential (ATP.(ADP.Psub(i))/sup -1/) and adenylate energy charge ((ATP + 0.5.ADP).(AdN)/sup -1/) are significantly reduced in damaged needles. This is due to relatively higher levels of Psub(i) and of AMP. The results, although incomplete and preliminary, indicate metabolic alterations which have been described for other tissues in response to pollution by photooxidants.

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.

Estructura del aparato reproductor masculino de Uca uruguayensis (Decapoda, Ocypodidae Structure of the male reproductive tract of Uca uruguayensis (Decapoda, Ocypodidae

Directory of Open Access Journals (Sweden)

Elena Irene Cuartas

Full Text Available El objetivo de este trabajo es describir las estructuras del sistema reproductor de machos de Uca uruguayensis Nobili, 1901 y estudiar los cambios relacionados con la madurez sexual. Se trabajó con observación estereoscópica de material fresco, técnica histológica de rutina y observaciones en Microscopio Electrónico de Barrido. Se incluyó la caracterización de los gonopodos 1 (G1 y 2 (G2. El tracto reproductor del macho se compone de un par de testículos (T, un vaso deferente (VD tubular y sinuoso y una ampolla terminal (AT. El VD tiene tres secciones, la anterior (VDA, la media (VDM y la posterior (VDP. El VDA y VDM están conformadas por un epitelio simple de células cúbicas. El epitelio del VDP es columnar y con núcleos basales y alongados. El diseño tubular se modifica al ocuparse el lumen del VDP con líquido espermático durante el verano y la musculatura circular se hace más evidente. La porción terminal del VDP se ensancha formando una ampolla (AT que comprende cuatro cámaras interconectadas. Todas las estructuras están rodeadas de una capa de tejido conectivo de poco espesor. Se identifican las modificaciones observadas en la histología de T y VD, definiendo como mas relevantes las observadas desde el mes de noviembre hasta marzo. Estas modificaciones sugieren que U. uruguayensis tiene, en la localidad estudiada, una única estación reproductiva durante el verano en esta latitud. La AT, tal como es descripta, es una estructura que hasta el momento no ha sido mencionada para los Brachyura.The structure of the male reproductive tract was described in Uca uruguayensis Nobili, 1901, by using histological methods, scanning electron microscopy techniques, and stereoscopic observations of fresh material. The aim of this work was to establish the functional changes associated with sexual maturation. The morphology of the first (G1 and second (G2 pair of gonopods was described. The male reproductive tract consists of paired

Switching speeds in NCAP displays: dependence on collection angle and wavelength

Science.gov (United States)

Reamey, Robert H.; Montoya, Wayne; Wartenberg, Mark

1991-06-01

The on and off switching speeds of nematic droplet-polymer films (NCAP) are shown to depend on the collection angle (f/#) and the wavelength of the light used in the measurement. Conventional twisted nematic liquid crystal displays have switching speeds which depend little on these factors. The switching speed dependence on collection angle (f/#) and wavelength for nematic droplet-polymer films is inherent to the mechanism by which light is modulated in these films. This mechanism is the scattering of light by the nematic droplets. The on times become faster and the off times become slower as the collection angle of detection is increased. The overall change in switching speed can be large. Greater than 100X changes in off time have been observed. As the wavelength of the light used to interrogate the sample is increased (blue yields green yields red) the on times become faster and the off times become slower. This dependence of switching speed on wavelength is apparent at all collection angles. An awareness of these effects is necessary when developing nematic droplet-polymer films for display applications and when comparing switching speed data from different sources.

Motility, ATP levels and metabolic enzyme activity of sperm from bluegill (Lepomis macrochirus).

Science.gov (United States)

Burness, Gary; Moyes, Christopher D; Montgomerie, Robert

2005-01-01

Male bluegill displays one of two life history tactics. Some males (termed "parentals") delay reproduction until ca. 7 years of age, at which time they build nests and actively courts females. Others mature precociously (sneakers) and obtain fertilizations by cuckolding parental males. In the current study, we studied the relations among sperm motility, ATP levels, and metabolic enzyme activity in parental and sneaker bluegill. In both reproductive tactics, sperm swimming speed and ATP levels declined in parallel over the first 60 s of motility. Although sneaker sperm initially had higher ATP levels than parental sperm, by approximately 30 s postactivation, no differences existed between tactics. No differences were noted between tactics in swimming speed, percent motility, or the activities of key metabolic enzymes, although sperm from parentals had a higher ratio of creatine phosphokinase (CPK) to citrate synthase (CS). In both tactics, with increasing CPK and CS activity, sperm ATP levels increased at 20 s postactivation, suggesting that capacities for phosphocreatine hydrolysis and aerobic metabolism may influence interindividual variation in rates of ATP depletion. Nonetheless, there was no relation between sperm ATP levels and either swimming speed or percent of sperm that were motile. This suggests that interindividual variation in ATP levels may not be the primary determinant of variation in sperm swimming performance in bluegill.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

Science.gov (United States)

Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

2015-07-03

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Influence of temperature on daily locomotor activity in the crab Uca pugilator.

Directory of Open Access Journals (Sweden)

Audrey M Mat

Full Text Available Animals living in the intertidal zone are exposed to prominent temperature changes. To cope with the energetic demands of environmental thermal challenges, ectotherms rely mainly on behavioral responses, which may change depending on the time of the day and seasonally. Here, we analyze how temperature shapes crabs' behavior at 2 different times of the year and show that a transition from constant cold (13.5°C to constant warm (17.5°C water temperature leads to increased locomotor activity levels throughout the day in fiddler crabs (Uca pugilator collected during the summer. In contrast, the same transition in environmental temperature leads to a decrease in the amplitude of the daily locomotor activity rhythm in crabs collected during the winter. In other words, colder temperatures during the cold season favor a more prominent diurnal behavior. We interpret this winter-summer difference in the response of daily locomotor activity to temperature changes within the framework of the circadian thermoenergetics hypothesis, which predicts that a less favorable energetic balance would promote a more diurnal activity pattern. During the winter, when the energetic balance is likely less favorable, crabs would save energy by being more active during the expected high-temperature phase of the day-light phase-and less during the expected low-temperature phase of the day-dark phase. Our results suggest that endogenous rhythms in intertidal ectotherms generate adaptive behavioral programs to cope with thermoregulatory demands of the intertidal habitat.

The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

Science.gov (United States)

Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

2015-05-22

HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

CO2-Induced ATP-Dependent Release of Acetylcholine on the Ventral Surface of the Medulla Oblongata.

Science.gov (United States)

Huckstepp, Robert T R; Llaudet, Enrique; Gourine, Alexander V

2016-01-01

Complex mechanisms that detect changes in brainstem parenchymal PCO2/[H+] and trigger adaptive changes in lung ventilation are responsible for central respiratory CO2 chemosensitivity. Previous studies of chemosensory signalling pathways suggest that at the level of the ventral surface of the medulla oblongata (VMS), CO2-induced changes in ventilation are (at least in part) mediated by the release and actions of ATP and/or acetylcholine (ACh). Here we performed simultaneous real-time biosensor recordings of CO2-induced ATP and ACh release from the VMS in vivo and in vitro, to test the hypothesis that central respiratory CO2 chemosensory transduction involves simultaneous recruitment of purinergic and cholinergic signalling pathways. In anaesthetised and artificially ventilated rats, an increase in inspired CO2 triggered ACh release on the VMS with a peak amplitude of ~5 μM. Release of ACh was only detected after the onset of CO2-induced activation of the respiratory activity and was markedly reduced (by ~70%) by ATP receptor blockade. In horizontal slices of the VMS, CO2-induced release of ATP was reliably detected, whereas CO2 or bath application of ATP (100 μM) failed to trigger release of ACh. These results suggest that during hypercapnia locally produced ATP induces or potentiates the release of ACh (likely from the medullary projections of distal groups of cholinergic neurones), which may also contribute to the development and/or maintenance of the ventilatory response to CO2.

ATP signals

DEFF Research Database (Denmark)

Novak, Ivana

2016-01-01

The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas......The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas...

Effects of simulated oil exposure on two intertidal macrozoo benthos: Tympanotonus fuscata (L.) and Uca tangeri (Eydoux, 1935) in a tropical estuarine ecosystem.

Science.gov (United States)

Ewa-Oboho, I O; Abby-Kalio, N J

1994-08-01

The impacts of simulated Nigerian light crude oil on mud flat periwinkles, Tympanotonus fuscata (L.), and fiddler crabs, Uca tangeri (Eydoux, 1935) was examined through field experiments conducted in the Bonny estuary of the Niger Delta (southern Nigeria). The purpose was to assess the fate and effects of a known quantity of the Nigerian light crude oil on this environment. Drastic changes in the densities of T. fuscata and U. tangeri observed immediately after spills was attributed to the effects of the oil. A large increase in Uca biomass occurred in the affected area. Salinity and temperature in the study area showed little fluctuations throughout the survey. Sediment characteristics were similar for all sites (stations). Grain-size analysis revealed that sediments at the study area were 70% silt. Migration of oil via tidal percolation was observed as much as 11 cm beneath the sediment surface.

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

Science.gov (United States)

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. © 2013 The Authors Journal compilation © 2013 FEBS.

The energetic state of mitochondria modulates complex III biogenesis through the ATP-dependent activity of Bcs1.

Science.gov (United States)

Ostojić, Jelena; Panozzo, Cristina; Lasserre, Jean-Paul; Nouet, Cécile; Courtin, Florence; Blancard, Corinne; di Rago, Jean-Paul; Dujardin, Geneviève

2013-10-01

Our understanding of the mechanisms involved in mitochondrial biogenesis has continuously expanded during the last decades, yet little is known about how they are modulated to optimize the functioning of mitochondria. Here, we show that mutations in the ATP binding domain of Bcs1, a chaperone involved in the assembly of complex III, can be rescued by mutations that decrease the ATP hydrolytic activity of the ATP synthase. Our results reveal a Bcs1-mediated control loop in which the biogenesis of complex III is modulated by the energy-transducing activity of mitochondria. Although ATP is well known as a regulator of a number of cellular activities, we show here that ATP can be also used to modulate the biogenesis of an enzyme by controlling a specific chaperone involved in its assembly. Our study further highlights the intramitochondrial adenine nucleotide pool as a potential target for the treatment of Bcs1-based disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

Gamma-radiation effect of the ATP-ASE-activity in various parts of cotton sprouts

International Nuclear Information System (INIS)

Kazimov, A.K.

1975-01-01

ATP-ase is a thiol enzyme whose sulfhydryl group plays an important role. The transport of substances through biological membranes is the result of the action of the sodium-potassium pump of the cell, which functions with ATP energy. The action of this transport mechanism depends on the activity of ATP-ase. It may be postulated, therefore, that the suppression of the active transport of Na + and K + ions in cells under irradiation is partially the result of a disturbance of the activity of the ATP enzyme system. The author studied the effect of gamma radiation on ATP-ase activity in various parts of seven-day-old seedlings of type 108-F cotton, which were irradiated using Co 60 gamma radiation. The results of the experiment showed that the ATP-ase activity of the cotton seedling rootlets depends on the dose and the time elapsed after irradiation (a table is given). Small radiation doses (0.2 and 0.5 krad) significantly increased ATP-ase activity in the rootlets, while heavy doses inhibited it significantly. Similar results were obtained for the stems and leaves (tables are given). It was estblished that the ATP-ase of cotton seedlings has varying sensitivity to irradiation. The most sensitive ATP-ases were those of the rootlets. The activity of background ATP-ase is less subject to change than Na + and K + activated ATP-ases. For example, while the activity of ATP-ase (without ions) was inhibited by 25% when a 25 krad irradiation dose was administered, the retardation of Na + and K + activated ATP-ases reached 41%. The author suggests that the inhibition of ATP-ase activity under irradiation is mainly the result of a disturbance of the structure of the membrane functions. It is also possible that ATP-ase activity decreases because of a lack of the enzyme substrate - ATP, which is formed during the process of oxydative phosphorylization. A table is also provided showing the effect of irradiation on the activity of ATP-ase activated by various ions in the roots of

The vibrational signals that male fiddler crabs ( Uca lactea) use to attract females into their burrows

Science.gov (United States)

Takeshita, Fumio; Murai, Minoru

2016-06-01

In some fiddler crab species, males emit vibrations from their burrows to mate-searching females after they have attracted a female to the burrow entrance using a waving display. Although the vibrations are considered acoustic signals to induce mating, it has not been demonstrated whether the vibrations attract the females into the burrow and, consequently, influence females' mating decisions. We investigated the structures and patterns of the vibrations using a dummy female and demonstrated experimentally a female preference for male vibrations in Uca lactea in the field. The acoustic signals consisted of repetitions of pulses. The dominant frequency of the pulses decreased with male carapace width. The pulse length decreased slightly with an increasing number of vibrational repetitions, and the pulse interval increased with increasing repetitions. These factors imply that the vibrations convey information on male characteristics, such as body size and stamina. In the experiment on female mate choice, the females significantly preferred males with higher pulse repetition rates when they were positioned at the entrance of the burrow, indicating that the females use the male vibrational signals to decide whether to enter the burrow. However, females showed no preference for the vibrations once they were inside a burrow, i.e., whether they decided to copulate, suggesting that the vibrations do not independently affect a female's final decision of mate choice. The vibrations inside the burrow might influence a female's decision by interaction with other male traits such as the burrow structure.

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

International Nuclear Information System (INIS)

Milgrom, Elena M.; Milgrom, Yakov M.

2012-01-01

Highlights: ► MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. ► V-ATPase activity saturation with MgATP is not sufficient for complete protection. ► The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K m values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers

NARCIS (Netherlands)

Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

2014-01-01

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned

Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity

Directory of Open Access Journals (Sweden)

Francesca Schena

2013-06-01

Full Text Available Immunoglobulin (Ig isotype diversification by class switch recombination (CSR is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID. Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures

Science.gov (United States)

Koizumi, Schuichi; Fujishita, Kayoko; Tsuda, Makoto; Shigemoto-Mogami, Yukari; Inoue, Kazuhide

2003-09-01

Originally ascribed passive roles in the CNS, astrocytes are now known to have an active role in the regulation of synaptic transmission. Neuronal activity can evoke Ca2+ transients in astrocytes, and Ca2+ transients in astrocytes can evoke changes in neuronal activity. The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between astrocytes and neurons. We demonstrate here that ATP, a primary mediator of intercellular Ca2+ signaling among astrocytes, also mediates intercellular signaling between astrocytes and neurons in hippocampal cultures. Mechanical stimulation of astrocytes evoked Ca2+ waves mediated by the release of ATP and the activation of P2 receptors. Mechanically evoked Ca2+ waves led to decreased excitatory glutamatergic synaptic transmission in an ATP-dependent manner. Exogenous application of ATP does not affect postsynaptic glutamatergic responses but decreased presynaptic exocytotic events. Finally, we show that astrocytes exhibit spontaneous Ca2+ waves mediated by extracellular ATP and that inhibition of these Ca2+ responses enhanced excitatory glutamatergic transmission. We therefore conclude that ATP released from astrocytes exerts tonic and activity-dependent down-regulation of synaptic transmission via presynaptic mechanisms.

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth

Energy Technology Data Exchange (ETDEWEB)

Ashino, T.; Varadarajan, S.; Urao, N.; Oshikawa, J.; Chen, G. -F.; Wang, H.; Huo, Y.; Finney, L.; Vogt, S.; McKinney, R. D.; Maryon, E. B.; Kaplan, J. H.; Ushio-Fukai, M.; Fukai, T. (Biosciences Division); ( XSD); ( PSC-USR); (Univ. of Illinois at Chicago); (Univ. of Minnesota)

2010-09-09

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

ATP secretion from nerve trunks and Schwann cells mediated by glutamate.

Science.gov (United States)

Liu, Guo Jun; Bennett, Max R

2003-11-14

ATP release from rat sciatic nerves and from cultured Schwann cells isolated from the nerves was investigated using an online bioluminescence technique. ATP was released in relatively large amounts from rat sciatic nerve trunks during electrical stimulation. This release was blocked by the sodium channel inhibitor tetrodotoxin and the non-NMDA glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Schwann cells isolated from the nerve trunks did not release ATP when electrically stimulated but did in response to glutamate in a concentration-dependent manner. Glutamate-stimulated ATP release was inhibited by specific non-competitive AMPA receptor antagonist GYKI 52466 and competitive non-NMDA receptor antagonist CNQX. Glutamate-stimulated ATP release was decreased by inhibition of anion transporter inhibitors by furosemide, cystic fibrosis transmembrane conductance regulator by glibenclamide and exocytosis by botulinum toxin A, indicating that anion transporters and exocytosis provide the main secretion mechanisms for ATP release from the Schwann cells.

Metabolism of ATP-binding cassette drug transporter inhibitors: complicating factor for multidrug resistance.

NARCIS (Netherlands)

Cnubben, N.H.; Wortelboer, H.M.; Zanden, J.J. van; Rietjens, I.M.; Bladeren, P.J. van

2005-01-01

Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

Science.gov (United States)

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

Science.gov (United States)

Droguett, Karla; Rios, Mariana; Carreño, Daniela V; Navarrete, Camilo; Fuentes, Christian; Villalón, Manuel; Barrera, Nelson P

2017-07-15

Extracellular ATP, in association with [Ca 2+ ] i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca 2+ ] i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca 2+ ] i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml -1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast

DEFF Research Database (Denmark)

Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D.

2014-01-01

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been...

ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB

International Nuclear Information System (INIS)

Koo, Hyeon-Sook; Liu, L.F.; Claassen, L.; Grossman, L.

1991-01-01

The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAM complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. The authors results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed

Physiology and pathophysiology of K(ATP) channels in the pancreas and cardiovascular system: a review.

Science.gov (United States)

Seino, Susumu

2003-01-01

K(ATP) channels are present in pancreatic and extrapancreatic tissues such as heart and smooth muscle, and display diverse molecular composition. They contain two different structural subunits: an inwardly rectifying potassium channel subunit (Kir6.x) and a sulfonylurea receptor (SURX). Recent studies on genetically engineered Kir6.2 knockout mice have provided a better understanding of the physiological and pathophysiological roles of Kir6.2-containing K(ATP) channels. Kir6.2/SUR1 has a pivotal role in pancreatic insulin secretion. Kir6.2/SUR2A mediates the effects of K(ATP) channels openers on cardiac excitability and contractility and contributes to ischemic preconditioning. However, controversy remains on the physiological properties of the K(ATP) channels in vascular smooth muscle cells. Kir6.1 knockout mice exhibit sudden cardiac death due to cardiac ischemia, indicating that Kir6.1 rather than Kir6.2 is critical in the regulation of vascular tone. This article summarizes current understanding of the physiology and pathophysiology of Kir6.1- and Kir6.2-containing K(ATP) channels.

ATP as a Multi-target Danger Signal in the Brain

Directory of Open Access Journals (Sweden)

Ricardo J Rodrigues

2015-04-01

Full Text Available ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR, which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection.

Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

Science.gov (United States)

Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

2014-11-01

Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

Optimum ratio of AET, ATP and serotonin applied in combinations determined with a reference to their toxicity

International Nuclear Information System (INIS)

Benova, D.K.; P''tev, I.Kh.

1985-01-01

In experiments on mice, a study was made of the quantitative dependence of toxicity of AET, ATP and serotonin applied in combinations. The toxicity decreased when ATP was combined with AET and increased when ATP of AET were combined with serotonin. The toxicity of a combination of all three substances was reduced by introducing high doses of ATP

ATP-dependent molecular chaperones in plastids--More complex than expected.

Science.gov (United States)

Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

2015-09-01

Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

Science.gov (United States)

Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E; Spurgeon, Harold A; Sollott, Steven J; Lakatta, Edward G

2011-11-01

In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. Published by Elsevier Ltd.

Quantal release of ATP from clusters of PC12 cells.

Science.gov (United States)

Fabbro, Alessandra; Skorinkin, Andrei; Grandolfo, Micaela; Nistri, Andrea; Giniatullin, Rashid

2004-10-15

Although ATP is important for intercellular communication, little is known about the mechanism of endogenous ATP release due to a dearth of suitable models. Using PC12 cells known to express the P2X2 subtype of ATP receptors and to store ATP with catecholamines inside dense-core vesicles, we found that clusters of PC12 cells cultured for 3-7 days generated small transient inward currents (STICs) after an inward current elicited by exogenous ATP. The amplitude of STICs in individual cells correlated with the peak amplitude of ATP-induced currents. STICs appeared as asynchronous responses (approximately 20 pA average amplitude) for 1-20 s and were investigated with a combination of patch clamping, Ca2+ imaging, biochemistry and electron microscopy. Comparable STICs were produced by focal KCl pulses and were dependent on extracellular Ca2+. STICs were abolished by the P2X antagonist PPADS and potentiated by Zn2+, suggesting they were mediated by P2X2 receptor activation. The highest probability of observing STICs was after the peak of intracellular Ca2+ increase caused by KCl. Biochemical measurements indicated that KCl application induced a significant release of ATP from PC12 cells. Electron microscopy studies showed narrow clefts without 'synaptic-like' densities between clustered cells. Our data suggest that STICs were caused by quantal release of endogenous ATP by depolarized PC12 cells in close juxtaposition to the recorded cell. Thus, STICs may be a new experimental model to characterize the physiology of vesicular release of ATP and to study the kinetics and pharmacology of P2X2 receptor-mediated quantal currents.

ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

Science.gov (United States)

Liao, Jielou

2004-03-01

Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

Direct demonstration of ATP-dependent release of SecA from a translocating preprotein by surface plasmon resonance

NARCIS (Netherlands)

de Keyzer, J; van der Does, C; Kloosterman, TG; Driessen, AJM

2003-01-01

Translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds with high affinity to the membrane-embedded protein-conducting SecYEG complex and serves as both a receptor for secretory proteins and as an ATP-driven molecular motor. Cycles of ATP binding

Photon energy dependence and angular response of glass display used in mobile phones for accident dosimetry

International Nuclear Information System (INIS)

Discher, Michael; Greiter, Matthias; Woda, Clemens

2014-01-01

Previous studies have shown that glass displays extracted from mobile phones are suitable as emergency dosimeters in case of an accidental radiation overexposure using the thermoluminescence (TL) method. So far these studies have focused only on recovering the absorbed dose to the material. However, dose in air or dose to the victim carrying the device might be significantly different. Therefore the aim of this work was to investigate photon energy dependence and angular response of glass display used in modern mobile phones. An over-response of about a factor of five is observed for low photon energies compared to the response to Cs-137 (662 keV) which is in reasonable agreement with calculated values mass energy-absorption coefficients of glass and air. Little variation in the energy dependence can be seen for glass displays coming from three different mobile phone models. The angular response for display glass is flat with regard to air kerma within the incident angle of ±60°, independent of the irradiation setup used (with a water phantom or with air kerma reference conditions). For incident angles of 90° the shielding effect of the mobile phones becomes important. With the dosimetric characterization of the photon energy and angular dependencies the absorbed dose in a glass display can be transferred to a reference air kerma dose and provides a useful option for retrospective accident dosimetry. - Highlights: • Determination of the photon energy dependence and angular response for display glass used as an accident dosimeter. • Over-response of about a factor of five for low photon energies. • Flat angular response within incident angles between ±60°

Effects and mechanism of acid rain on plant chloroplast ATP synthase.

Science.gov (United States)

Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-09-01

Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity.

Science.gov (United States)

Chalat, Madhavan; Moleschi, Kody; Molday, Robert S

2017-02-01

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2. © 2017 Chalat et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

ATP and MO25alpha regulate the conformational state of the STRADalpha pseudokinase and activation of the LKB1 tumour suppressor.

Directory of Open Access Journals (Sweden)

Elton Zeqiraj

2009-06-01

Full Text Available Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADalpha binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADalpha for MO25alpha, and conversely, binding of MO25alpha promotes interaction of STRADalpha with ATP. Mutagenesis studies reveal that association of STRADalpha with either ATP or MO25alpha is essential for LKB1 activation. We conclude that ATP and MO25alpha cooperate to maintain STRADalpha in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADalpha that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE syndrome. We demonstrate this mutation destabilizes STRADalpha and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADalpha to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25alpha binding. Thus, the function of STRADalpha is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

NARCIS (Netherlands)

Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.

2015-01-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential

Increased ATP generation in the host cell is required for efficient vaccinia virus production

Directory of Open Access Journals (Sweden)

Hsu Che-Fang

2009-09-01

Full Text Available Abstract To search for cellular genes up-regulated by vaccinia virus (VV infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

Antispasmodic activity of Symplocos paniculata is mediated through opening of ATP-dependent K+ channel

Directory of Open Access Journals (Sweden)

Khalid Hussain Janbaz

2016-06-01

Full Text Available Symplocos paniculata is a medicinal plant used by native healers to manage gastrointestinal ailments. The crude methanolic extract of S. paniculata was screened pharmacologically both in vitro and in vivo for the validation of its therapeutic potential. It suppressed the spontaneous activity of isolated rabbit jejunum preparations and also caused inhibition of the low K+ (20 mM- induced spastic contractions in isolated rabbit jejunum preparations in a manner comparable to cromakalim. The relaxant effect was found to be blocked following glibenclamide exposure of the isolated tissue preparations similar to cromakalim, suggesting that observed response was likely to be mediated through opening of ATP dependent K+ channels. Following oral administration to mice provided protection against castor oil-induced diarrhea in a manner similar to loperamide. The plant material was found safe in toxicity study up to oral dose of 8 g/kg in mice. Hence, present study provides a scientific basis for the vernacular use of S. paniculata in gastro-intestinal system.

BAD-dependent regulation of fuel metabolism and K(ATP) channel activity confers resistance to epileptic seizures.

Science.gov (United States)

Giménez-Cassina, Alfredo; Martínez-François, Juan Ramón; Fisher, Jill K; Szlyk, Benjamin; Polak, Klaudia; Wiwczar, Jessica; Tanner, Geoffrey R; Lutas, Andrew; Yellen, Gary; Danial, Nika N

2012-05-24

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional K(ATP) channels in the rat retinal microvasculature: topographical distribution, redox regulation, spermine modulation and diabetic alteration.

Science.gov (United States)

Ishizaki, Eisuke; Fukumoto, Masanori; Puro, Donald G

2009-05-15

The essential task of the circulatory system is to match blood flow to local metabolic demand. However, much remains to be learned about this process. To better understand how local perfusion is regulated, we focused on the functional organization of the retinal microvasculature, which is particularly well adapted for the local control of perfusion. Here, we assessed the distribution and regulation of functional K(ATP) channels whose activation mediates the hyperpolarization induced by adenosine. Using microvascular complexes freshly isolated from the rat retina, we found a topographical heterogeneity in the distribution of functional K(ATP) channels; capillaries generate most of the K(ATP) current. The initiation of K(ATP)-induced responses in the capillaries supports the concept that the regulation of retinal perfusion is highly decentralized. Additional study revealed that microvascular K(ATP) channels are redox sensitive, with oxidants increasing their activity. Furthermore, the oxidant-mediated activation of these channels is driven by the polyamine spermine, whose catabolism produces oxidants. In addition, our observation that spermine-dependent oxidation occurs predominately in the capillaries accounts for why they generate most of the K(ATP) current detected in retinal microvascular complexes. Here, we also analysed retinal microvessels of streptozotocin-injected rats. We found that soon after the onset of diabetes, an increase in spermine-dependent oxidation at proximal microvascular sites boosts their K(ATP) current and thereby virtually eliminates the topographical heterogeneity of functional K(ATP) channels. We conclude that spermine-dependent oxidation is a previously unrecognized mechanism by which this polyamine modulates ion channels; in addition to a physiological role, spermine-dependent oxidation may also contribute to microvascular dysfunction in the diabetic retina.

Single K ATP channel opening in response to action potential firing in mouse dentate granule neurons.

Science.gov (United States)

Tanner, Geoffrey R; Lutas, Andrew; Martínez-François, Juan Ramón; Yellen, Gary

2011-06-08

ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

[The 2,3-diphosphoglycerate shunt and stabilization of the ATP level in mammalian erythrocytes].

Science.gov (United States)

Ataullakhanov, A I; Ataullakhanov, F I; Vitvitskiĭ, V M; Zhabotinskiĭ, A M; Pichugin, A V

1985-06-01

The mechanisms of regulation of energy metabolism in erythrocytes of various mammalian species were investigated. In native erythrocytes of man, sheep, cow, dog and mouse the dependencies of the rates of glucose uptake on ATP concentration (i.e., regulatory parameters of glycolysis) were measured. These parameters plotted in normalized coordinates are not species-specific (invariant). The dependence of the rate of ATP-consuming processes on ATP concentration has been studied for the first time in intact mammalian erythrocytes. This dependence was found to be linear only in the species, in whose erythrocytes the activity of 2,3-diphosphoglycerate shunt is practically zero. In all species under study, the stabilization of ATP level is provided for mainly by the hexokinase-phosphofructokinase system. A comparison of regulatory mechanisms of energy metabolism in mammalian (sheep, cow) erythrocytes, in which the 2,3-diphosphoglycerate shunt is absent, with human and animal erythrocytes, in which this pathway is active, points to the important role of the 2,3-diphosphoglycerate shunt in regulation of energy conversion in erythrocytes. This shunt operates as an additional stabilizer protecting the cell from extremal influences.

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

Energy Technology Data Exchange (ETDEWEB)

Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

1988-07-12

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) 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 Mg/sup 2 +/ 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 CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. 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 (/sup 32/P)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 Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ 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 Ca/sup 2 +/ as the activating cation.

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

Directory of Open Access Journals (Sweden)

Juan Francisco Codocedo

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

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 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......, respectively (Pcontracting muscle (Pmuscle, whereas interstitial NE concentrations increased similarly in both active...... and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial prinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its...

A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia.

Science.gov (United States)

Fu, Liang-Wu; Longhurst, John C

2010-12-01

Myocardial ischemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Brief myocardial ischemia leads to ATP release in the interstitial space. Furthermore, exogenous ATP and α,β-methylene ATP (α,β-meATP), a P2X receptor agonist, stimulate cutaneous group III and IV sensory nerve fibers. The present study tested the hypothesis that endogenous ATP excites cardiac afferents during ischemia through activation of P2 receptors. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) in anesthetized cats. Single fields of 45 afferents (conduction velocities = 0.25-4.92 m/s) were identified in the left ventricle with a stimulating electrode. Five minutes of myocardial ischemia stimulated 39 of 45 cardiac afferents (8 Aδ, 37 C fibers). Epicardial application of ATP (1-4 μmol) stimulated six ischemically sensitive cardiac afferents in a dose-dependent manner. Additionally, epicardial ATP (2 μmol), ADP (2 μmol), a P2Y agonist, and α,β-meATP (0.5 μmol) significantly activated eight other ischemically sensitive afferents. Third, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist, abolished the responses of six afferents to epicardial ATP (2 μmol) and attenuated the ischemia-related increase in activity of seven other afferents by 37%. In the absence of P2 receptor blockade, cardiac afferents responded consistently to repeated application of ATP (n = 6) and to recurrent myocardial ischemia (n = 6). Finally, six ischemia-insensitive cardiac spinal afferents did not respond to epicardial ATP (2-4 μmol), although these afferents did respond to epicardial bradykinin. Taken together, these data indicate that, during ischemia, endogenously released ATP activates ischemia-sensitive, but not ischemia-insensitive, cardiac spinal afferents through stimulation of P2 receptors likely located on the cardiac sensory

Content dependent selection of image enhancement parameters for mobile displays

Science.gov (United States)

Lee, Yoon-Gyoo; Kang, Yoo-Jin; Kim, Han-Eol; Kim, Ka-Hee; Kim, Choon-Woo

2011-01-01

Mobile devices such as cellular phones and portable multimedia player with capability of playing terrestrial digital multimedia broadcasting (T-DMB) contents have been introduced into consumer market. In this paper, content dependent image quality enhancement method for sharpness and colorfulness and noise reduction is presented to improve perceived image quality on mobile displays. Human visual experiments are performed to analyze viewers' preference. Relationship between the objective measures and the optimal values of image control parameters are modeled by simple lookup tables based on the results of human visual experiments. Content dependent values of image control parameters are determined based on the calculated measures and predetermined lookup tables. Experimental results indicate that dynamic selection of image control parameters yields better image quality.

The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

Science.gov (United States)

Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

2009-01-01

Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release.

Science.gov (United States)

Forsyth, Alison M; Wan, Jiandi; Owrutsky, Philip D; Abkarian, Manouk; Stone, Howard A

2011-07-05

RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold.

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

Science.gov (United States)

Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

2017-08-01

Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

Directory of Open Access Journals (Sweden)

Masaki Nakano

2017-08-01

Full Text Available Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances, which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

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

Science.gov (United States)

Brand, M D; Lehninger, A L

1977-01-01

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

Greater ATP dependence than sodium dependence of radiocalcium efflux in bullfrog ventricle

International Nuclear Information System (INIS)

Brommundt, G.; Kavaler, F.

1985-01-01

45 Ca efflux was studied in intact bullfrog ventricles following a 2-h period of loading with radiocalcium-containing Ringer solution. The cannulated ventricle was placed in a closed air-filled container to which were applied rhythmic, electronically timed, positive- and negative-pressure pulsations, which induced ventricular volume excursions. The mechanical arrangement and timing circuitry made it possible for each period to be as short in duration as 15 s. By use of this technique, penetration of the extracellular space by [ 14 C]inulin was found to be complete within 30 s, and recovery of the inulin proceeded with a time constant of 17-24 s, indicating a completeness of recovery of 98% within 90 s. Washout of added 45 Ca was quantitatively quite close to that of inulin, and in addition the estimated rate of sequestration of the isotope was slow enough to introduce only a small error into the experimental results. 45 Ca efflux was only slightly (15%) sensitive to replacement of extracellular sodium but was profoundly sensitive to the inhibitors of ATP synthesis, cyanide and 2-deoxy-glucose

Journey in guidelines for lipid management: From adult treatment panel (ATP-I to ATP-III and what to expect in ATP-IV

Directory of Open Access Journals (Sweden)

P G Talwalkar

2013-01-01

Full Text Available Adult Treatment Panel (ATP, an expert panel to supervise cholesterol management was set up under the aegis of National Cholesterol Education Program (NCEP in 1985. Since then NCEP-ATP has been revising and framing guidelines to enable clinician to deliver better treatment to cardiovascular patients and to educate general people. As a result, considerable reduction in cardiovascular related deaths has been observed in recent times. All three ATP guidelines viz. ATP-I, ATP-II and ATP-III have targeted low density lipoprotein as their primary goal. The ATP-III guideline was updated in the light of evidences from 5-major clinical trials and was released in 2004. It added therapeutic lifestyle changes, concept of risk equivalents, Framingham CHD-risk score non-high density lipoprotein cholesterol (non-HDL-C as secondary target and gave strong emphasis on metabolic risk factors. The earlier treat-to-target paradigm faced fierce criticism from clinicians across the globe because of insufficient proof of safety and benefits of treating patients with respect to an individual′s low density lipoprotein (LDL level. Further, demonstration of non-HDL-C and total cholesterol/HDL-C ratio as strong predictors of overall cardiovascular risk foresees new guidelines. A tailored-treatment approach was suggested instead of LDL-C target based treatment approach which was soundly based on direct clinical trials evidences and proposes treatment based on individual′s overall 5- to 10-year cardiovascular risk irrespective of LDL-C level, leading to lower number of people on high dose/s of statins. Recent report of the Cholesterol Treatment Trialist′s Collaborators meta-analysis strongly supported primary prevention of LDL with statins in low risk individuals and showed that its benefits completely outweighed its known hazards. Markers other than LDL-C like apolipoprotein B, non-HDL-C and total cholesterol/HDL-C ratio would take precedence in the risk assessment and

Ca2+ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

Science.gov (United States)

Lee, Jaekwang; Chun, Ye-Eun; Han, Kyung-Seok; Lee, Jungmoo; Woo, Dong Ho

2015-01-01

Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of Gαq-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte. PMID:25792866

Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa.

Science.gov (United States)

Yi, Young-Joo; Sutovsky, Miriam; Kennedy, Chelsey; Sutovsky, Peter

2012-01-01

Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.

Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa.

Directory of Open Access Journals (Sweden)

Young-Joo Yi

Full Text Available Inorganic pyrophosphate (PPi is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1 in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.

ATP-sensitive K(+-channels in muscle cells: features and physiological role

Directory of Open Access Journals (Sweden)

O. B. Vadzyuk

2014-08-01

Full Text Available ATP-sensitive K+-channels of plasma membranes belong to the inward rectifier potassium channels type. They are involved in coupling of electrical activity of muscle cell with its metabolic­ state. These channels are heterooctameric and consist of two types of subunits: four poreforming (Kir 6.х and four regulatory (SUR, sulfonylurea receptor. The Kir subunits contain highly selective K+ filter and provide for high-velocity K+ currents. The SUR subunits contain binding sites for activators and blockers and have metabolic sensor, which enables channel activation under conditions of metabolic stress. ATP blocks K+ currents through the ATP-sensitive K+-channels in the most types of muscle cells. However, functional activity of these channels does not depend on absolute concentration of ATP but on the АТР/ADP ratio and presence of Mg2+. Physiologically active substances, such as phosphatidylinositol bisphosphate and fatty acid esters can regulate the activity of these structures in muscle cells. Activation of these channels under ischemic conditions underlies their cytoprotective action, which results in prevention of Ca2+ overload in cytosol. In contrast to ATP-sensitive K+-channels of plasma membranes, the data regarding the structure and function of ATP-sensitive K+-channels of mitochondrial membrane are contradictory. Pore-forming subunits of this channel have not been firmly identified yet. ATP-sensitive K+ transport through the mitochondrial­ membrane is easily tested by different methods, which are briefly reviewed in this paper. Interaction of mitoKATP with physiological and pharmacological ligands is discussed as well.

Direct ATP photolabeling of Escherichia coli recA proteins: identification of regions required for ATP binding

International Nuclear Information System (INIS)

Banks, G.R.; Sedgwick, S.G.

1986-01-01

When the Escherichia coli RecA protein is UV irradiated in the presence of [alpha- 32 P]ATP, a labeled protein--ATP adduct is formed. All the experimental evidence indicates that, in forming such an adduct, the ATP becomes specifically immobilized in the catalytically relevant ATP binding site. The adduct can also be identified after irradiation of E. coli cell lysates in a similar manner. This direct ATP photolabeling of RecA proteins has been used to identify regions of the polypeptide chain involved in the binding of ATP. The photolabeling of a RecA protein that lacks wild-type carboxy-terminal amino acids is not detectable. A RecA protein in which the amino-terminal sequence NH2-Ala-Ile-Asp-Glu-Asn- is replaced by NH2-Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser- is only about 5% as efficiently photolabeled as the wild-type protein. Both of these RecA protein constructions, however, contain all the elements previously implicated, directly or indirectly, in the binding of ATP. ATP-photolabeled RecA protein has also been chemically cleaved at specific amino acids in order to identify regions of the polypeptide chain to which the nucleotide becomes covalently photolinked. The evidence is consistent with a region comprising amino acids 116-170. Thus, this work and that of others suggest that several disparate regions of the unfolded polypeptide chain may combine to form the ATP binding site upon protein folding or may influence binding through long-range effects

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

Directory of Open Access Journals (Sweden)

Ronglan Zhao

Full Text Available 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.

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter.

Science.gov (United States)

Bao, Huan; Dalal, Kush; Cytrynbaum, Eric; Duong, Franck

2015-10-16

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease.

Science.gov (United States)

Takaya, Akiko; Tabuchi, Fumiaki; Tsuchiya, Hiroko; Isogai, Emiko; Yamamoto, Tomoko

2008-06-01

Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

Wind power communication design and implementation of test environment for IEC61850/UCA2

Energy Technology Data Exchange (ETDEWEB)

Johnsson, A.; Svensson, J.

2002-04-01

Elforsk has sponsored a joint Swedish-Danish project aiming at finding and recommend a common solution for communication with wind power plants. The first stage of the work resulted in a requirement specification Functional Requirements on Communication System for Wind Turbine Applications. During the project a number of possible communication solutions were identified. The two most promising solutions have been tested in order to verify to what extent they fulfil the requirements in the specification. A version of the IEC 61850 standard based on the communication protocol MMS, has been tested at a wind power plant at Gotland, Sweden, and an OPC-interface has been tested in Denmark. This report includes a description of the design choices made for the test implementation of MMS, as well as a detailed description of the implementation of the IEC 61850/UCA2 software including information models and information exchange services. (BA)

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

Science.gov (United States)

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

Characterization of cellular protective effects of ATP13A2/PARK9 expression and alterations resulting from pathogenic mutants.

Science.gov (United States)

Covy, Jason P; Waxman, Elisa A; Giasson, Benoit I

2012-12-01

Mutations in ATP13A2, which encodes a lysosomal P-type ATPase of unknown function, cause an autosomal recessive parkinsonian syndrome. With mammalian cells, we show that ATP13A2 expression protects against manganese and nickel toxicity, in addition to proteasomal, mitochondrial, and oxidative stress. Consistent with a recessive mode of inheritance of gene defects, disease-causing mutations F182L and G504R are prone to misfolding and do not protect against manganese and nickel toxicity because they are unstable as a result of degradation via the endoplasmic reticulum-associated degradation (ERAD)-proteasome system. The protective effects of ATP13A2 expression are not due to inhibition of apoptotic pathways or a reduction in typical stress pathways, insofar as these pathways are still activated in challenged ATP13A2-expressing cells; however, these cells display a dramatic reduction in the accumulation of oxidized and damaged proteins. These data indicate that, contrary to a previous suggestion, ATP13A2 is unlikely to convey cellular resilience simply by acting as a lysosomal manganese transporter. Consistent with the recent identification of an ATP13A2 recessive mutation in Tibetan terriers that develop neurodegeneration with neuronal ceroid lipofucinoses, our data suggest that ATP13A2 may function to import a cofactor required for the function of a lysosome enzyme(s). Copyright © 2012 Wiley Periodicals, Inc.

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...

A conserved residue cluster that governs kinetics of ATP-dependent gating of Kir6.2 potassium channels

DEFF Research Database (Denmark)

Zhang, Roger S; Wright, Jordan; Pless, Stephan Alexander

2015-01-01

modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp68, Lys170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp68...... or Lys170 markedly slow the kinetics of channel opening (500 ms and 700 ms for Trp68Leu and Lys170Asn, respectively), while increasing channel open probability. Examining the functional effects of these residues using phi-value analysis revealed a steep negative slope. This finding implies...

ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

Science.gov (United States)

Liu, Jun; Liu, Wenjing; Yang, Jun

2016-02-11

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress.

Science.gov (United States)

Vassilopoulos, Athanassios; Pennington, J Daniel; Andresson, Thorkell; Rees, David M; Bosley, Allen D; Fearnley, Ian M; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Deng, Chu-Xia; Walker, John E; Gius, David

2014-08-01

Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

ATP-binding cassette (ABC) transporters in normal and pathological lung

NARCIS (Netherlands)

van der Deen, M; de Vries, EGE; Timens, W; Scheper, RJ; Timmer-Bosscha, H; Postma, DS

2005-01-01

ATP-binding cassette ( ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein ( P-gp), multidrug resistance-associated protein 1 ( MRP1) and

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

Directory of Open Access Journals (Sweden)

Anthony eFord

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.

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

DEFF Research Database (Denmark)

Gourdon, P.; Sitsel, Oleg; Karlsen, J.L.

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......, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type...

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

Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition.

Science.gov (United States)

Woo, Joohan; Jun, Young Keul; Zhang, Yin-Hua; Nam, Joo Hyun; Shin, Dong Hoon; Kim, Sung Joon

2018-02-01

TWIK-related two-pore domain K + channels (TREKs) are regulated by intracellular pH (pH i ) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Previously, Glu 306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pH i -sensing residue. The direction of PI(4,5)P 2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P 2 formation. Here we investigate the anionic and cationic residues of pCt for the pH i and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (I TREK-2,i-o ), acidic pH i -induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pH i sensitivity of I TREK-2,i-o . Unlike the inhibition of wild-type (WT) I TREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P 2 (10 μM) abolished I TREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P 2 . In whole-cell recordings of TREK-2 (I TREK-2,w-c ), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pH e 6.9. I TREK-1,w-c of WT is largely suppressed by pH e 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pH i sensitivity.

Effect of mercury on survival, metabolism and behavior of larval Uca pugilator (Brachyura)

Energy Technology Data Exchange (ETDEWEB)

DeCoursey, P J; Vernberg, W B

1972-01-01

A battery of tests was used to determine the effects of three dilute mercuric chloride solutions on larval stages (Zoea I, III, V) of the fiddler crab Uca pugilator(Bosc). The influence of both acute and chronic exposures on viability, oxygen consumption, and swimming activity was measured. No stage V and only a few stage I or III larvae were able to survive a concentration of 9 x 10/sup -7/ M HgCl/sub 2/ (0.18 ppm Hg) longer than 24 hr; an exposure as short as 6 hr resulted in reduced metabolism and swimming rate of all stages. Although concentrations of 9 x 10/sup -9/ M Hg Cl/sub 2/ (0.0018 ppm) and 9 x 10/sup -11/ M HgCl/sub 2/ (0.000018 ppm) were sublethal, 24-hr exposures did affect metabolism and swimming. Some larvae reared in the more dilute mercury solutions developed to the megalopa stage, but survival was reduced in relation to the mercury concentration. The data from all tests suggest that toxicity of mercury increases with larval age. 20 references, 6 figures.

Effect of Intramuscular Protons, Lactate, and ATP on Muscle Hyperalgesia in Rats.

Directory of Open Access Journals (Sweden)

Nicholas S Gregory

Full Text Available Chronic muscle pain is a significant health problem leading to disability[1]. Muscle fatigue can exacerbate muscle pain. Metabolites, including ATP, lactate, and protons, are released during fatiguing exercise and produce pain in humans. These substances directly activate purinergic (P2X and acid sensing ion channels (ASICs on muscle nociceptors, and when combined, produce a greater increase in neuron firing than when given alone. Whether the enhanced effect of combining protons, lactate, and ATP is the sum of individual effects (additive or more than the sum of individual effects (synergistic is unknown. Using a rat model of muscle nociceptive behavior, we tested each of these compounds individually over a range of physiologic and supra-physiologic concentrations. Further, we combined all three compounds in a series of dilutions and tested their effect on muscle nociceptive behavior. We also tested a non-hydrolyzable form of ATP (α,β-meATP alone and in combination with lactate and acidic pH. Surprisingly, we found no dose-dependent effect on muscle nociceptive behavior for protons, lactate, or ATP when given alone. We similarly found no effect after application of each two-metabolite combination. Only pH 4 saline and α,β-meATP produced hyperalgesia when given alone. When all 3 substances were combined, however, ATP (2.4μm, lactate (10mM, and acidic pH (pH 6.0 produced an enhanced effect greater than the sum of the effects of the individual components, i.e. synergism. α,β me ATP (3nmol, on the other hand, showed no enhanced effects when combined with lactate (10mM or acidic pH (pH 6.0, i.e. additive. These data suggest that combining fatigue metabolites in muscle produces a synergistic effect on muscle nociception.

Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

Science.gov (United States)

Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

1996-01-01

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

Effectiveness of onsite wastewater reuse system in reducing bacterial contaminants measured with human-specific IMS/ATP and qPCR.

Science.gov (United States)

Agidi, Senyo; Vedachalam, Sridhar; Mancl, Karen; Lee, Jiyoung

2013-01-30

Water shortages and the drive to recycle is increasing interest in reuse of reclaimed wastewater. Timely and cost-effective ways to detect fecal pollutants prior to reuse increases confidence of residents and neighbors concerned about reuse of reclaimed wastewater. The on-site wastewater treatment and reuse systems (OWTRS) used in this study include a septic tank, peat bioreactor, ClO(2) disinfection and land spray irrigation system. Bacteroides fragilis, Escherichia coli and Enterococcus spp., were tested with immunomagnetic separation/ATP bioluminescence (IMS/ATP), qPCR and culture-based methods. The results displayed a 2-log reduction in fecal bacteria in the peat bioreactor and a 5-log reduction following chloride dioxide disinfection. The fecal bacteria levels measured by IMS/ATP correlated with qPCR results: HuBac 16S (R(2) = 0.903), Bf-group 16S (R(2) = 0.956), gyrB (R(2) = 0.673), and Ent 23S (R(2) = 0.724). This is the first study in which the newly developed human-specific IMS/ATP and previously developed IMS/ATP were applied for determining OWTRS efficiency. Results of the study revealed that IMS/ATP is a timely and cost-effective way to detect fecal contaminants, and results were validated with qPCR and culture based methods. The new IMS/ATP can also be applied broadly in the detection of human-originated fecal contamination. Copyright © 2012 Elsevier Ltd. All rights reserved.

Keragaman Genetik Sekuen Gen ATP Synthase FO Subunit 6 (ATP6 Monyet Hantu (Tarsius Indonesia (GENETIC DIVERSITY STUDY OF ATP6 GENE SEQUENCES OF TARSIERS FROM INDONESIA

Directory of Open Access Journals (Sweden)

Rini Widayanti

2013-07-01

Full Text Available In a conservation effort, the identification of Tarsier species, on the bases of the morphological andmolecular characteristic is necessary. Up to now, the identification of the animals were based on themorphology and vocalizations, which is extremely difficult to identify each, tarsier species. The objective ofthis research was to study the genetic diversity on ATP6 gene of Tarsius sp. Based on sequencing of PCRproduct using primer ATP6F and ATP6R with 681 nts. PCR product. The sequence of ATP6 fragmentswere aligned with other primates from Gene bank with aid of software Clustal W, and were analyzed usingMEGA program version 4.0. Three different nucleotide sites were found (nucleotide no. 288, 321 and 367.The genetic distance based on nucleotide ATP6 sequence calculated using Kimura 2-parameter modelindicated that the smallest genetic distance 0%, biggest 0.8% and average 0, 2%. The phylogenetic treeusing neighbor joining method based on the sequence of nucleotide ATP6 gene could not be used todifferentiate among T. Dianae (from Central Sulawesi, T. Spectrum (from North Sulawesi, T. bancanus(from lampung, South Sumatera and T.bancanus from West Kalimantan.

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

Science.gov (United States)

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Nomata, Jiro [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601 (Japan); Terauchi, Kazuki [Department of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577 (Japan); Fujita, Yuichi, E-mail: fujita@agr.nagoya-u.ac.jp [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601 (Japan)

2016-02-12

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN–BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e{sup –}) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratio of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase. - Highlights: • The stoichiometry of nitrogenase-like protochlorophyllide reductase was determined. • The minimal ATP/2e{sup –} ratio was 4, which coincides with that of nitrogenase. • The ATP/2e{sup –} ratio increases with increasing L-protein/NB-protein molar ratio. • DPOR has an intrinsic property, but retains features shared with nitrogenase.

Acidic pH facilitates peripheral αβmeATP-mediated nociception in rats: differential roles of P2X, P2Y, ASIC and TRPV1 receptors in ATP-induced mechanical allodynia and thermal hyperalgesia.

Science.gov (United States)

Seo, Hyoung-Sig; Roh, Dae-Hyun; Kwon, Soon-Gu; Yoon, Seo-Yeon; Kang, Suk-Yun; Moon, Ji-Young; Choi, Sheu-Ran; Beitz, Alvin J; Lee, Jang-Hern

2011-03-01

Peripheral ischemia is commonly associated with an increase in tissue ATP concentration and a decrease in tissue pH. Although in vitro data suggest that low tissue pH can affect ATP-binding affinities to P2 receptors, the mechanistic relationship between ATP and low pH on peripheral nociception has not been fully examined. This study was designed to investigate the potential role of an acidified environment on intraplantar αβmeATP-induced peripheral pain responses in rats. The mechanical allodynia (MA) produced by injection of αβmeATP was significantly increased in animals that received the drug diluted in pH 4.0 saline compared to those that received the drug diluted in pH 7.0 saline. Moreover, animals injected with αβmeATP (100 nmol) in pH 4.0 saline developed thermal hyperalgesia (TH), which did not occur in animals treated with αβmeATP diluted in pH 7.0 saline. To elucidate which receptors were involved in this pH-related facilitation of αβmeATP-induced MA and TH, rats were pretreated with PPADS (P2 antagonist), TNP-ATP (P2X antagonist), MRS2179 (P2Y1 antagonist), AMG9810 (TRPV1 antagonist) or amiloride (ASIC blocker). Both PPADS and TNP-ATP dose-dependently blocked pH-facilitated MA, while TH was significantly reduced by pre-treatment with MRS2179 or AMG9810. Moreover, amiloride injection significantly reduced low pH-induced facilitation of αβmeATP-mediated MA, but not TH. These results demonstrate that low tissue pH facilitates ATP-mediated MA via the activation of P2X receptors and ASICs, whereas TH induced by ATP under low pH conditions is mediated by the P2Y1 receptor and TRPV1, but not ASIC. Thus distinct mechanisms are responsible for the development of MA and TH under conditions of tissue acidosis and increased ATP. Copyright © 2010 Elsevier Ltd. All rights reserved.

Color dependence with horizontal-viewing angle and colorimetric characterization of two displays using different backlighting

Science.gov (United States)

Castro, José J.; Pozo, Antonio M.; Rubiño, Manuel

2013-11-01

In this work we studied the color dependence with a horizontal-viewing angle and colorimetric characterization of two liquid-crystal displays (LCD) using two different backlighting: Cold Cathode Fluorescent Lamps (CCFLs) and light-emitting diodes (LEDs). The LCDs studied had identical resolution, size, and technology (TFT - thin film transistor). The colorimetric measurements were made with the spectroradiometer SpectraScan PR-650 following the procedure recommended in the European guideline EN 61747-6. For each display, we measured at the centre of the screen the chromaticity coordinates at horizontal viewing angles of 0, 20, 40, 60 and 80 degrees for the achromatic (A), red (R), green (G) and blue (B) channels. Results showed a greater color-gamut area for the display with LED backlight, compared with the CCFL backlight, showing a greater range of colors perceptible by human vision. This color-gamut area diminished with viewing angle for both displays. Higher differences between trends for viewing angles were observed in the LED-backlight, especially for the R- and G-channels, demonstrating a higher variability of the chromaticity coordinates with viewing angle. The best additivity was reached by the LED-backlight display (a lower error percentage). LED-backlight display provided better color performance of visualization.

ATP Binding cassette transporter gene expression in rat liver progenitor cells

NARCIS (Netherlands)

Ros, J.E.; Roskams, T.A.D.; Geuken, M.; Havinga, R.; Splinter, P.L.; Petersen, B.E.; LaRusso, N.F.; Kolk, van der D.M.; Kuipers, F.; Faber, K.N.; Müller, M.R.; Jansen, P.L.M.

2003-01-01

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

ATP binding cassette transporter gene expression in rat liver progenitor cells

NARCIS (Netherlands)

Ros, J. E.; Roskams, T. A. D.; Geuken, M.; Havinga, R.; Splinter, P. L.; Petersen, B. E.; LaRusso, N. F.; van der Kolk, D. M.; Kuipers, F.; Faber, K. N.; Müller, M.; Jansen, P. L. M.

2003-01-01

BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

ATP binding cassette transporter gene expression in rat liver progenitor cells

NARCIS (Netherlands)

Ros, J.E.; Roskams, TAD; Geuken, M; Havinga, R; Splinter, PL; Petersen, BE; LaRusso, NF; van der Kolk, D.M.; Kuipers, F; Faber, KN; Muller, M; Jansen, PLM

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

Anticancer ruthenium(III) complex KP1019 interferes with ATP-dependent Ca2+ translocation by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA).

Science.gov (United States)

Sadafi, Fabrizio-Zagros; Massai, Lara; Bartolommei, Gianluca; Moncelli, Maria Rosa; Messori, Luigi; Tadini-Buoninsegni, Francesco

2014-08-01

Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a P-type ATPase that sustains Ca2+ transport and plays a major role in intracellular Ca2+ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium-based anticancer drugs, namely KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]), NAMI-A (imidazolium [trans-tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)]) and RAPTA-C ([Ru(η6-p-cymene)dichloro(1,3,5-triaza-7-phosphaadamantane)]), and cisplatin (cis-diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid-supported membrane was measured after ATP or Ca2+ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP-dependent translocation of Ca2+ ions. An IC50 value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca2+ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Duchenne muscular dystrophy: normal ATP turnover in cultured cells

International Nuclear Information System (INIS)

Fox, I.H.; Bertorini, T.; Palmieri, G.M.A.; Shefner, R.

1986-01-01

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

Participatory Methods and UCA Project: understanding technologies as culture

Directory of Open Access Journals (Sweden)

Magda Pischetola

2015-12-01

Full Text Available Abstract In the complex and changing context of digital culture, the media become an important space of relation, as they have the crucial role of articulating new cultural logics that lead to disruptions in the school environment. To understand this change, new methods of analysis and research have been created, the so-called Participatory Methodologies. They are action research strategies aimed at intervening in a given social situation. In the analysis proposed here, such methodologies will help us to address the challenge of involving digital technologies in school culture, through the participation of different individuals involved. Two qualitative case studies about the project Um Computador por Aluno – the Brazilian One Laptop per Child -, carried out in 2012 in the schools of Santa Catarina and Bahia, are the first of two phases of the research presented. The results concern a "vertical" form of technology insertion in schools, which led to frustration and de-motivation at several levels. Starting from these considerations, the second stage of research proposes a pedagogical intervention in one of four schools in the field. The methodologies of participatory video and photography are chosen as possibilities of action-reflection-action on the sociocultural reality of students through the experience of sharing. The results show the importance of carrying out creative activities, appropriate to a social conception of learning, as well as the centrality of children and youth as agency and a broader need to redefine the relationship between teacher and student, in a more "horizontal" perspective process of teaching and learning. Keywords: Projeto UCA. Participatory Research Method. Innovative teaching-learning.

Searing sentiment or cold calculation? the effects of leader emotional displays on team performance depend on follower epistemic motivation

NARCIS (Netherlands)

van Kleef, G.A.; Homan, A.C.; Beersma, B.; van Knippenberg, D.; van Knippenberg, B.; Damen, F.

2009-01-01

We examined how leader emotional displays affect team performance. We developed and tested the idea that effects of leader displays of anger versus happiness depend on followers' epistemic motivation, which is the desire to develop a thorough understanding of a situation. Experimental data on

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

Science.gov (United States)

Pateraki, Irini; Renato, Marta; Azcón-Bieto, Joaquín; Boronat, Albert

2013-04-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active 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 synthesize ATP de novo. We propose that the replacement of the γ-subunit present in tomato leaf and green fruit chloroplasts by the atypical γ-subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

A taste for ATP: neurotransmission in taste buds

Science.gov (United States)

Kinnamon, Sue C.; Finger, Thomas E.

2013-01-01

Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat, and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells. PMID:24385952

A taste for ATP: neurotransmission in taste buds

Directory of Open Access Journals (Sweden)

Thomas E. Finger

2013-12-01

Full Text Available Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells.

Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

DEFF Research Database (Denmark)

Skjørringe, Tina; Pedersen, Per Amstrup; Thorborg, Sidsel Salling

2017-01-01

Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher...

Energy-dependent imaging in digital radiography: a review on acquisition, processing and display technique

International Nuclear Information System (INIS)

Coppini, G.; Maltinti, G.; Valli, G.; Baroni, M.; Buchignan, M.; Valli, G.

1986-01-01

The capabilities of energy-dependent imaging in digital radiography are analyzed paying particular attention to digital video systems. The main techniques developed in recent years for selective energy imaging are reviewed following a unified approach. Discussion about advantages and limits of energy methods is carried out by a comparative analysis of computer simulated data and experimental results as obtained by standard x-ray equipments coupled to a digital video unit. Geometric phantoms are used as test object, as also images of a chest phantom are produced. Since signal-to-noise ratio degradation is one of the major problems when dealing with selective imaging, a particular effort is made to investigate noise effects. In this perspective, an original colour encoding display of energy sequences is presented. By mapping the various energy measurements on different colour bands (typically those of an RGB TV-monitor), an increased image conspicuity is obtained without a significant noise degradation: this is ensured by the energy dependence of attenuation coefficients and by the integrating characteristics of the display device

Extracellular ATP induces albuminuria in pregnant rats

NARCIS (Netherlands)

Faas, M.M.; van der Schaaf, G.; Borghuis, T.; Jongman, R.M.; van Pampus, Maria; de Vos, P.; van Goor, Harry; Bakker, W.W.

BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula.

Optimisation of ATP determination in drinking water

DEFF Research Database (Denmark)

Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

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

A novel de novo mutation in ATP1A3 and childhood-onset schizophrenia

Science.gov (United States)

Smedemark-Margulies, Niklas; Brownstein, Catherine A.; Vargas, Sigella; Tembulkar, Sahil K.; Towne, Meghan C.; Shi, Jiahai; Gonzalez-Cuevas, Elisa; Liu, Kevin X.; Bilguvar, Kaya; Kleiman, Robin J.; Han, Min-Joon; Torres, Alcy; Berry, Gerard T.; Yu, Timothy W.; Beggs, Alan H.; Agrawal, Pankaj B.; Gonzalez-Heydrich, Joseph

2016-01-01

We describe a child with onset of command auditory hallucinations and behavioral regression at 6 yr of age in the context of longer standing selective mutism, aggression, and mild motor delays. His genetic evaluation included chromosomal microarray analysis and whole-exome sequencing. Sequencing revealed a previously unreported heterozygous de novo mutation c.385G>A in ATP1A3, predicted to result in a p.V129M amino acid change. This gene codes for a neuron-specific isoform of the catalytic α-subunit of the ATP-dependent transmembrane sodium–potassium pump. Heterozygous mutations in this gene have been reported as causing both sporadic and inherited forms of alternating hemiplegia of childhood and rapid-onset dystonia parkinsonism. We discuss the literature on phenotypes associated with known variants in ATP1A3, examine past functional studies of the role of ATP1A3 in neuronal function, and describe a novel clinical presentation associated with mutation of this gene. PMID:27626066

Detection of microbial contaminations in drinking water using ATP measurements – evaluating potential for online monitoring

DEFF Research Database (Denmark)

Vang, Óluva Karin; Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

2011-01-01

There is an increasing call for fast and reliable methods for continuous monitoring of microbial drinking water quality in order to protect public health. The potential for Adenosine triphosphate (ATP) measurements as a real-time analysis for continuous monitoring of microbial drinking water...... quality was investigated through simulation of two contamination scenarios, i.e. drinking water contaminated with waste water and surface water at various concentrations. With ATP measurements it was possible to detect waste water diluted 1000-10,000 times in drinking water depending on sensitivity...... of reagent kit. Surface water diluted 100-1000 times was detected in drinking water with ATP measurements. ATP has the potential as an early warning tool, especially in the period when the contamination concentration is high. 2011 © American Water Works Association AWWA WQTC Conference Proceedings All Rights...

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

KAUST Repository

Jourdain, P.

2016-02-19

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

KAUST Repository

Jourdain, P.; Allaman, I.; Rothenfusser, K.; Fiumelli, Hubert; Marquet, P.; Magistretti, Pierre J.

2016-01-01

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

Effect of ATP sulfurylase overexpression in bright yellow 2 tobacco cells: regulation of ATP sulfurylase and SO4(-2) transport activities

International Nuclear Information System (INIS)

Hatzfeld, Y.; Cathala, N.; Grignon, C.; Davidian, J.C.

1998-01-01

To determine if the ATP sulfurylase reaction is a regulatory step for the SO4(2-)-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 355 promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO4(2-) influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO4(2-) (a toxic SO4(2-) analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

Science.gov (United States)

Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

2011-10-01

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

Science.gov (United States)

Sumi, Daigo; Asao, Masashi; Okada, Hideta; Yogi, Kuniko; Miyataka, Hideki; Himeno, Seiichiro

2016-06-01

Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity.

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

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active...... 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...

Characterization of a Ca2+ response to both UTP and ATP at human P2Y11 receptors: evidence for agonist-specific signaling.

Science.gov (United States)

White, Pamela J; Webb, Tania E; Boarder, Michael R

2003-06-01

Previous reports on heterologously-expressed human P2Y11 receptors have indicated that ATP, but not UTP, is an agonist stimulating both phosphoinositidase C and adenylyl cyclase. Consistent with these findings, we report that in 1321N1 cells expressing human P2Y11 receptors, UTP stimulation did not lead to accumulation of inositol(poly)phosphates under conditions in which ATP gave a robust, concentration-dependent effect. Unexpectedly, however, both UTP and ATP stimulated increases in cytosolic Ca2+ concentration ([Ca2+]c), with both nucleotides achieving similar EC50 and maximal responses. The responses to maximally effective concentrations of ATP and UTP were not additive. The [Ca2+]c increase in response to UTP was less dependent on extracellular Ca2+ than was the response to ATP. AR-C67085 (2-propylthio-beta,gamma-difluoromethylene-d-ATP, a P2Y11-selective agonist), adenosine 5'-O-(3-thiotriphosphate), and benzoyl ATP were all full agonists with potencies similar to those of ATP and UTP. In desensitization experiments, exposure to ATP resulted in loss of the UTP response; this response was more sensitive to desensitization than that of ATP. Pertussis toxin pretreatment attenuated the response to UTP but left the ATP response unaffected. The presence of 2-aminoethyl diphenylborate differentially affected the responses of ATP and UTP. No mRNA transcripts for P2Y2 or P2Y4 were detectable in the P2Y11-expressing cells. We conclude that UTP is a Ca2+-mobilizing agonist at P2Y11 receptors and that ATP and UTP acting at the same receptor recruit distinct signaling pathways. This example of agonist-specific signaling is discussed in terms of agonist trafficking and differential signal strength.

Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

Energy Technology Data Exchange (ETDEWEB)

Gruenhagen, Jason Alan [Iowa State Univ., Ames, IA (United States)

2003-01-01

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. Ca²⁺ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca²⁺ 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⁺ 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

Comparação da estrutura populacional das espécies de Uca (Crustacea: Decapoda: Ocypodidae) no Manguezal de Itacuruçá, Rio de Janeiro, Brasil Comparison of the population structure of the species of Uca (Crustacea: Decapoda: Ocypodidae) in the mangrove of Itacuruçá, Rio Janeiro, Brazil

OpenAIRE

Luciane M. Bedê; Lídia M. Y. Oshiro; Luziane M. D. Mendes; Alessandra A. Silva

2008-01-01

Este trabalho foi realizado no Manguezal de Itacuruçá, na Baía de Sepetiba com o objetivo de analisar a estrutura populacional das espécies de Uca Leach, 1814. Foram realizadas coletas de junho/2005 a maio/2006, durante as marés baixas. Os caranguejos foram capturados manualmente por duas pessoas e durante 15 minutos. Um total de 2580 animais foi coletado, sendo 1465 machos e 1115 fêmeas. Com relação ao tamanho dos indivíduos, observou-se que os animais do Manguezal de Itacuruçá, de maneira g...

Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

Science.gov (United States)

Carlier, M F; Criquet, P; Pantaloni, D; Korn, E D

1986-02-15

Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

Science.gov (United States)

Gomes, P; Chevalier, J; Boesmans, W; Roosen, L; van den Abbeel, V; Neunlist, M; Tack, J; Vanden Berghe, P

2009-08-01

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

The mitochondrial phosphate transporters modulate plant responses to salt stress via affecting ATP and gibberellin metabolism in Arabidopsis thaliana.

Directory of Open Access Journals (Sweden)

Wei Zhu

Full Text Available The mitochondrial phosphate transporter (MPT plays crucial roles in ATP production in plant cells. Three MPT genes have been identified in Arabidopsis thaliana. Here we report that the mRNA accumulations of AtMPTs were up-regulated by high salinity stress in A. thaliana seedlings. And the transgenic lines overexpressing AtMPTs displayed increased sensitivity to salt stress compared with the wild-type plants during seed germination and seedling establishment stages. ATP content and energy charge was higher in overexpressing plants than those in wild-type A. thaliana under salt stress. Accordingly, the salt-sensitive phenotype of overexpressing plants was recovered after the exogenous application of atractyloside due to the change of ATP content. Interestingly, Genevestigator survey and qRT-PCR analysis indicated a large number of genes, including those related to gibberellin synthesis could be regulated by the energy availability change under stress conditions in A. thaliana. Moreover, the exogenous application of uniconazole to overexpressing lines showed that gibberellin homeostasis was disturbed in the overexpressors. Our studies reveal a possible link between the ATP content mediated by AtMPTs and gibberellin metabolism in responses to high salinity stress in A. thaliana.

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...

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

DEFF Research Database (Denmark)

Jorgensen, N R; Geist, S T; Civitelli, R

1997-01-01

mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein......Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium...... stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

Science.gov (United States)

Pissarek, M; Garcia de Arriba, S; Schäfer, M; Sieler, D; Nieber, K; Illes, P

1998-10-01

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri

Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

Science.gov (United States)

Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

2013-12-01

One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

ATP-Dependent C–F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen

Directory of Open Access Journals (Sweden)

Oliver Tiedt

2016-08-01

Full Text Available Complete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2 and HF in the denitrifying Thauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA to benzoyl-coenzyme A (BzCoA and HF, catalyzed by class I BzCoA reductase (BCR. Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA.

Relationship between cavitation and loss of echogenicity from ultrasound contrast agents

OpenAIRE

Radhakrishnan, Kirthi; Bader, Kenneth B; Haworth, Kevin J; Kopechek, Jonathan A; Raymond, Jason L; Huang, Shao-Ling; McPherson, David D; Holland, Christy K

2013-01-01

Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pulse-duration dependent pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation. Previous studies have demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds ...

Selective decrease of components of the creatine kinase system and ATP synthase complex in chronic Chagas disease cardiomyopathy.

Directory of Open Access Journals (Sweden)

Priscila Camillo Teixeira

2011-06-01

Full Text Available BACKGROUND: Chronic Chagas disease cardiomyopathy (CCC is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC and ischemic (IC cardiomyopathies. METHODOLOGY/PRINCIPAL FINDINGS: Myocardium homogenates from CCC (N=5, IC (N=5 and IDC (N=5 patients, as well as from heart donors (N=5 were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit and muscular creatine kinase (CKM and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. CONCLUSIONS/SIGNIFICANCE: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

Science.gov (United States)

Belcik, J Todd; Davidson, Brian P; Xie, Aris; Wu, Melinda D; Yadava, Mrinal; Qi, Yue; Liang, Sherry; Chon, Chae Ryung; Ammi, Azzdine Y; Field, Joshua; Harmann, Leanne; Chilian, William M; Linden, Joel; Lindner, Jonathan R

2017-03-28

Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×10 8 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K ATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of

α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9).

Science.gov (United States)

Daniel, Guillaume; Musso, Alessandra; Tsika, Elpida; Fiser, Aris; Glauser, Liliane; Pletnikova, Olga; Schneider, Bernard L; Moore, Darren J

2015-01-01

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD. Copyright © 2015 Elsevier Inc. All rights reserved.

Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

International Nuclear Information System (INIS)

Zhang Yun-Xin

2014-01-01

Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis. (interdisciplinary physics and related areas of science and technology)

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

Science.gov (United States)

Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

2016-12-01

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Genetic variation in ATP5O is associated with skeletal muscle ATP50 mRNA expression and glucose uptake in young twins.

Directory of Open Access Journals (Sweden)

Tina Rönn

Full Text Available BACKGROUND: Impaired oxidative capacity of skeletal muscle mitochondria contribute to insulin resistance and type 2 diabetes (T2D. Furthermore, mRNA expression of genes involved in oxidative phosphorylation, including ATP5O, is reduced in skeletal muscle from T2D patients. Our aims were to investigate mechanisms regulating ATP5O expression in skeletal muscle and association with glucose metabolism, and the relationship between ATP5O single nucleotide polymorphisms (SNPs and risk of T2D. METHODOLOGY/PRINCIPAL FINDINGS: ATP5O mRNA expression was analyzed in skeletal muscle from young (n = 86 and elderly (n = 68 non-diabetic twins before and after a hyperinsulinemic euglycemic clamp. 11 SNPs from the ATP5O locus were genotyped in the twins and a T2D case-control cohort (n = 1466. DNA methylation of the ATP5O promoter was analyzed in twins (n = 22 using bisulfite sequencing. The mRNA level of ATP5O in skeletal muscle was reduced in elderly compared with young twins, both during basal and insulin-stimulated conditions (p<0.0005. The degree of DNA methylation around the transcription start of ATP5O was <1% in both young and elderly twins and not associated with mRNA expression (p = 0.32. The mRNA level of ATP5O in skeletal muscle was positively related to insulin-stimulated glucose uptake (regression coefficient = 6.6; p = 0.02. Furthermore, two SNPs were associated with both ATP5O mRNA expression (rs12482697: T/T versus T/G; p = 0.02 and rs11088262: A/A versus A/G; p = 0.004 and glucose uptake (rs11088262: A/A versus A/G; p = 0.002 and rs12482697: T/T versus T/G; p = 0.005 in the young twins. However, we could not detect any genetic association with T2D. CONCLUSIONS/SIGNIFICANCE: Genetic variation and age are associated with skeletal muscle ATP5O mRNA expression and glucose disposal rate, suggesting that combinations of genetic and non-genetic factors may cause the reduced expression of ATP5O in T2D muscle. These findings propose a role for ATP5O, in

Identification of a new Mpl-interacting protein, Atp5d.

Science.gov (United States)

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

2014-06-01

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

Binding of ATP by pertussis toxin and isolated toxin subunits

International Nuclear Information System (INIS)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-01-01

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

Binding of ATP by pertussis toxin and isolated toxin subunits

Energy Technology Data Exchange (ETDEWEB)

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

1990-07-03

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

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function

Directory of Open Access Journals (Sweden)

Wen-Jung Lu

2018-03-01

Full Text Available Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein, as well as cells lacking the outer membrane factor (OMF TolC (Tolerance to colicins. Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV, however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

Carbon and energy metabolism of atp mutants of Escherichia coli

DEFF Research Database (Denmark)

Jensen, Peter Ruhdal; Michelsen, Ole

1992-01-01

strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming......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...

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

Energy Technology Data Exchange (ETDEWEB)

Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

2009-01-01

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

Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

KAUST Repository

Beke-Somfai, T.; Lincoln, P.; Norden, B.

2013-01-01

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

KAUST Repository

Beke-Somfai, T.

2013-01-23

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

Science.gov (United States)

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

Respiratory ATP cost and benefit of arbuscular mycorrhizal symbiosis with Nicotiana tabacum at different growth stages and under salinity.

Science.gov (United States)

Del-Saz, Néstor Fernández; Romero-Munar, Antonia; Alonso, David; Aroca, Ricardo; Baraza, Elena; Flexas, Jaume; Ribas-Carbo, Miquel

2017-11-01

Growth and maintenance partly depend on both respiration and ATP production during oxidative phosphorylation in leaves. Under stress, ATP is needed to maintain the accumulated biomass. ATP production mostly proceeds from the cytochrome oxidase pathway (COP), while respiration via the alternative oxidase pathway (AOP) may decrease the production of ATP per oxygen consumed, especially under phosphorus (P) limitation and salinity conditions. Symbiosis with arbuscular mycorrhizal (AM) fungi is reputed by their positive effect on plant growth under stress at mature stages of colonization; however, fungal colonization may decrease plant growth at early stages. Thus, the present research is based on the hypothesis that AM fungus colonization will increase both foliar respiration and ATP production at mature stages of plant growth while decreasing them both at early stages. We used the oxygen-isotope-fractionation technique to study the in vivo respiratory activities and ATP production of the COP and AOP in AM and non-AM (NM) tobacco plants grown under P-limiting and saline conditions in sand at different growth stages (14, 28 and 49days). Our results suggest that AM symbiosis represents an ATP cost detrimental for shoot growth at early stages, whilst it represents a benefit on ATP allowing for faster rates of growth at mature stages, even under salinity conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Science.gov (United States)

Rasooli-Nejad, Seyed; Andrew, Jemma; Haydon, Philip G.; Pankratov, Yuriy

2014-01-01

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca2+-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca2+-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca2+ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca2+ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca2+-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes

Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex.

Directory of Open Access Journals (Sweden)

Ulyana Lalo

2014-01-01

Full Text Available Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca(2+-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca(2+-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using "sniff-cell" approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca(2+ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca(2+ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca(2+-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of

Properties of the manganese(II) binding site in ternary complexes of Mnter dot ADP and Mnter dot ATP with chloroplast coupling factor 1: Magnetic field dependence of solvent sup 1 H and sup 2 H NMR relaxation rates

Energy Technology Data Exchange (ETDEWEB)

Haddy, A.E.; Frasch, W.D.; Sharp, R.R. (Univ. of Michigan, Ann Arbor (USA))

1989-05-02

The influence of the binding of ADP and ATP on the high-affinity Mn(II) binding site of chloroplast coupling factor 1 (CF{sub 1}) was studied by analysis of field-dependent solvent proton and deuteron spin-lattice relaxation data. In order to characterize metal-nucleotide complexes of CF{sub 1} under conditions similar to those of the NMR experiments, the enzyme was analyzed for bound nucleotides and Mn(II) after incubation with AdN and MnCl{sub 2} and removal of labile ligands by extensive gel filtration chromatography. In the field-dependent NMR experiments, the Mn(II) binding site of CF{sub 1} was studied for three mole ratios of added Mn(II) to CF{sub 1}, 0.5, 1.0, and 1.5, in the presence of an excess of either ADP or ATP. The results were extrapolated to zero Mn(II) concentration to characterize the environment of the first Mn(II) binding site of Cf{sub 1}. In the presence of both adenine nucleotides, pronounced changes in the Mn(II) environment relative to that in Mn(II)-CF{sub 1} were evident; the local relaxation rate maxima were more pronounced and shifted to higher field strengths, and the relaxation rate per bound Mn(II) increased at all field strengths. Analysis of the data revealed that the number of exchangeable water molecules liganded to bound Mn(II) increased from one in the binary Mn(II)-CF{sub 1} complex to three and two in the ternary Mn(II)-ADP-CF{sub 1} and Mn(II)-ATP-CF{sub 1} complexes, respectively; these results suggest that a water ligand to bound Mn(II) in the Mn(II)-ADP-CF{sub 1} complex is replaced by the {gamma}-phosphate of ATP in the Mn(II)-ATP-CF{sub 1} complex. A binding model is presented to account for these observations.

Book Display as Adult Service

Directory of Open Access Journals (Sweden)

Matthew S. Moore

1997-03-01

Full Text Available 無Book display as an adult service is defined as choosing and positioning adult books from the collection to increase their circulation. The author contrasts bookstore arrangement for sales versus library arrangement for access. The paper considers the library-as-a-whole as a display, examines the right size for an in-library display, and discusses mass displays, end-caps, on-shelf displays, and the Tiffany approach. The author proposes that an effective display depends on an imaginative, unifying theme, and that book displays are part of the joy of libraries.

Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.

Science.gov (United States)

Yamasaki, Takashi; Nakazaki, Yosuke; Yoshida, Masasuke; Watanabe, Yo-hei

2011-07-01

ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer. © 2011 The Authors Journal compilation © 2011 FEBS.

Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

Science.gov (United States)

Ahn, Jinhi; Beharry, Seelochan; Molday, Laurie L; Molday, Robert S

2003-10-10

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

ATP7B detoxifies silver in ciliated airway epithelial cells

International Nuclear Information System (INIS)

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

2010-01-01

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

Sunscreens with broad-spectrum absorption decrease the trans TO cis photoisomerization of urocanic acid in the human stratum corneum after multiple UV light exposures

International Nuclear Information System (INIS)

Krien, P.M.; Moyal, D.

1994-01-01

The trans to cis photoisomerization of urocanic acid (UCA) in skin is considered to play an important role in the mechanism of immunosuppression. We have investigated the effects of skin type and various sunscreens with low sun protection factor (SPF) on the UV-induced cis-UCA formation in human skin after exposure to artificial UV light. The rate of cis-UCA formation depends little on the skin type and is reduced by topical application of sunscreens. The rate of cis-UCA formation decreases with increasing SPF and only broad-spectrum, highly protective sunscreens offer protection against the UV-induced formation of cis-UCA, which accumulates in the stratum corneum after multiple UV exposures. A theoretical approach to estimate the distribution of cis-UCA after irradiation indicates that this compound may diffuse into the deeper layers of the epidermis with D ∼ 10 -17 m 2 /s, and that its elimination from the stratum corneum is mainly due to desquamation. (author)

Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts

International Nuclear Information System (INIS)

Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

2014-01-01

Highlights: • Uniaxial stretching activates Ca 2+ signaling in human lung fibroblasts. • Stretch-induced intracellular Ca 2+ elevation is mainly via Ca 2+ influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca 2+ influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca 2+ ] i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca 2+ ] i . The stretch-induced [Ca 2+ ] i elevation was attenuated in Ca 2+ -free solution. In contrast, the increase of [Ca 2+ ] i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd 3+ , ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca 2+ ] i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca 2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP

Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex.

Science.gov (United States)

Su'etsugu, Masayuki; Shimuta, Toh-Ru; Ishida, Takuma; Kawakami, Hironori; Katayama, Tsutomu

2005-02-25

In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the beta-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg(168) in the AAA(+) Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain approximately 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA(+) proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA(+) domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

Energy Technology Data Exchange (ETDEWEB)

Sierra, Ana, E-mail: ana-sierra@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Subbotina, Ekaterina, E-mail: ekaterina-subbotina@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Zhu, Zhiyong, E-mail: zhiyong-zhu@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Gao, Zhan, E-mail: zhan-gao@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Koganti, Siva Rama Krishna, E-mail: sivaramakrishna.koganti@ttuhc.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Coetzee, William A., E-mail: william.coetzee@nyumc.org [Department of Pediatrics, NYU School of Medicine, New York, NY 10016 (United States); Goldhamer, David J., E-mail: david.goldhamer@uconn.edu [Center for Regenerative Biology, Department of Molecular and Cell Biology, Advanced Technology Laboratory, University of Connecticut, 1392 Storrs Road Unit 4243, Storrs, Connecticut 06269 (United States); Hodgson-Zingman, Denice M., E-mail: denice-zingman@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine, Iowa City, IA 52242 (United States); Zingman, Leonid V., E-mail: leonid-zingman@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Veterans Affairs, Medical Center, Iowa City, IA 52242 (United States)

2016-02-26

Sarcolemmal ATP-sensitive potassium (K{sub ATP}) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle K{sub ATP} channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle K{sub ATP} channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of K{sub ATP} channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish K{sub ATP} channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities. - Highlights: • ATP-sensitive K{sup +} channels regulate musclin production by skeletal muscles. • Lipolytic ANP signaling is promoted by augmented skeletal muscle musclin production. • Skeletal muscle musclin transcription is promoted by a CaMKII/HDAC/FOXO1 pathway. • Musclin links adipose mobilization to energy use in K{sub ATP} channel deficient skeletal muscle.

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

Science.gov (United States)

Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

2013-01-01

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

Modification of synthesis nucleotides [γ-32P] ATP

International Nuclear Information System (INIS)

Wira Y Rahman; Endang Sarmini; Herlina; Triyanto; Hambali; Abdul Mutalib; Santi Nurbaiti

2013-01-01

In molecular biology, radionuclides in the form of radiolabeled compounds have been widely used as deoxyribonucleic acid (DNA) / ribonucleic acid (RNA) tracer in order to explore a wide range of physiological and pathological processes. One of such compounds is [γ- 32 P]-adenosine triphosphate {[γ- 32 P]-ATP} [γ- 32 P]-ATP which has been widely used in the biotechnology research. In order to support the biotechnology research in Indonesia in this project, [γ- 32 P]- ATP had been synthesized by enzymatic reactions with modifying the method of synthesis using the precursor DL-glyceraldehyde 3-phosphate, nucleotides Adenosine Diphosphate (ADP) and H 3 32 PO 4 and enzymes glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoroglyceric phosphokinase and lactate dehydrogenase. The purification of the synthesized [γ- 32 P]-ATP, by using DEAE Sephadex column chromatography. The synthesis and purification process that had been performed were able in producing of [γ- 32 P]-ATP with radioactivity of 1,175 mCi and radiochemical purity of 99,49%.. Having successfully prepared the [γ- 32 P]-ATP and application, in the near future the Radioisotopes and Radiopharmaceuticals Centre is expected to be able in providing the above-mentioned radiolabeled nucleotide for biotechnology research in Indonesia. (author)

The Role of ATP in the Regulation of NCAM Function

DEFF Research Database (Denmark)

Hübschmann, Martin; Skladchikova, Galina

2008-01-01

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...... shedding, possibly affecting the structural plasticity associated with learning and memory....

Renal epithelial cells can release ATP by vesicular fusion

Directory of Open Access Journals (Sweden)

Randi G Bjaelde

2013-09-01

Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

Sildenafil protects neuronal cells from mitochondrial toxicity induced by β-amyloid peptide via ATP-sensitive K+ channels.

Science.gov (United States)

Son, Yonghae; Kim, Koanhoi; Cho, Hyok-Rae

2018-06-02

To understand the molecular mechanisms underlying the beneficial effects of sildenafil in animal models of neurological disorders, we investigated the effects of sildenafil on the mitochondrial toxicity induced by β-amyloid (Aβ) peptide. Treatment of HT-22 hippocampal neuronal cells with Aβ 25∼35 results in increased mitochondrial Ca 2+ load, which is subsequently suppressed by sildenafil as well as by diazoxide, a selective opener of the ATP-sensitive K + channels (K ATP ). However, the suppressive effects of sildenafil and diazoxide are significantly attenuated by 5-hydroxydecanoic acid (5-HD), a K ATP inhibitor. The increased mitochondrial Ca 2+ overload is accompanied by decrease in the intracellular ATP concentration, increase in intracellular ROS generation, occurrence of mitochondrial permeability transition, and activation of caspase-9 and cell death. Exposure to sildenafil inhibited the mitochondria-associated changes and cell death induced by Aβ. However, the inhibitory effects of sildenafil are abolished or weakened in the presence of 5-HD, suggesting that opening of the mitochondrial K ATP is required for sildenafil to exert these effects. Taken together, these results indicate that at the mitochondrial levels, sildenafil plays a protective role towards neuronal cell in an environment rich in Aβ, and exerts its effects via the mitochondrial K ATP channels-dependent mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

NCEP ATP III dan Framingham score

OpenAIRE

Hasan, Refli; Fahila, Reny

2016-01-01

Laporan ini merupakan Program Pendidikan Kolesterol National yang diperbaharui yaitu pedoman klinis untuk melakukan pengujian kolesterol dan manajemen. ATP III dibuat berdasarkan bukti dan laporan ekstensif yang akan menjadi referensi dan rekomendasi ilmiah. Laporan ATP III dapat dijadikan pedoman untuk pemberian terapi penurun kolesterol yang intensif dalam praktek. Pedoman ini hanya sebagai informasi , tidak dapat mempengaruhi secara mutlak dalam penilaian klinis dokter yang akhirnya menent...

Spatial distribution of fiddler crabs (Genus Uca in a tropical mangrove of northeast Brazil

Directory of Open Access Journals (Sweden)

Luis Ernesto Arruda Bezerra

2006-12-01

Full Text Available The influence of abiotic factors on the spatial distribution of the fiddler crabs Uca leptodactyla, U. maracoani, U. rapax and U. thayeri was studied in a tropical mangrove of northeast Brazil. Eight transects were delimited in a mangrove area of the Pacoti River. On each transect, three 0.25 m2 squares were sampled during spring low tide periods from September 2003 to August 2004. The sediment of the squares at each transect was analysed for grain size, organic matter and humidity. Morphology of the second maxilliped was studied considering the number of spoon-tipped setae to help explain the ecological distribution of the species. U. leptodactyla and U. rapax were found living in medium sand, U. thayeri was collected in fine and very fine sand, while U. maracoani was found living in very fine sand. U. leptodactyla showed a negative correlation with organic matter and humidity while U. thayeri showed a positive correlations for both factors. U. maracoani and U. rapax were not correlated with organic matter or humidity. The analysis of the second maxilliped revealed that U. leptodactyla and U. rapax show a high quantity of spoon-tipped setae while U. maracoani and U. thayeri show a greater quantity of plumose setae.

Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

Science.gov (United States)

Anwar, Zubeya; Albert, Jennifer L; Gubby, Sharon E; Boyle, John P; Roberts, Jonathon A; Webb, Tania E; Boarder, Michael R

1999-01-01

In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists.These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors. PMID:10510459

ATP-induced changes in rat skeletal muscle contractility.

Science.gov (United States)

Gabdrakhmanov, A I; Khayrullin, A E; Grishin, C H; Ziganshin, A U

2015-01-01

Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be

Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

NARCIS (Netherlands)

Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are

Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

International Nuclear Information System (INIS)

Kalayda, Ganna V; Wagner, Christina H; Buß, Irina; Reedijk, Jan; Jaehde, Ulrich

2008-01-01

Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant. In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn

Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

Directory of Open Access Journals (Sweden)

Reedijk Jan

2008-06-01

Full Text Available Abstract Background Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Methods Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of Results In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Conclusion Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours

Determination of glucose deficiency-induced cell death by mitochondrial ATP generation-driven proton homeostasis

Institute of Scientific and Technical Information of China (English)

Yanfen Cui; Yuanyuan Wang; Miao Liu; Li Qiu; Pan Xing; Xin Wang; Guoguang Ying; Binghui Li

2017-01-01

Glucose is one of major nutrients and its catabolism provides energy and/or building bricks for cell proliferation.Glucose deficiency results in cell death.However,the underlying mechanism still remains elusive.By using our recently developed method to monitor real-time cellular apoptosis and necrosis,we show that glucose deprivation can directly elicit necrosis,which is promoted by mitochondrial impairment,depending on mitochondrial adenosine triphosphate (ATP) generation instead of ATP depletion.We demonstrate that glucose metabolism is the major source to produce protons.Glucose deficiency leads to lack of proton provision while mitochondrial electron transfer chain continues consuming protons to generate energy,which provokes a compensatory iysosomal proton effiux and resultant increased lysosomal pH.This lysosomal alkalinization can trigger apoptosis or necrosis depending on the extent of alkalinization.Taken together,our results build up a metabolic connection between glycolysis,mitochondrion,and lysosome,and reveal an essential role of glucose metabolism in maintaining proton homeostasis to support cell survival.

Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

Directory of Open Access Journals (Sweden)

Blewett Ann

2008-12-01

Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

International Nuclear Information System (INIS)

Lodato, D.T.; Reed, G.H.

1987-01-01

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 17 O, 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 17 O regiospecifically incorporated into the γ-phosphate group of ATP. By contrast, 17 O in the α-phosphate or in the β-phosphate groups of ATP does not influence the spectrum. Experiments in 17 O-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

Performance of NCAP projection displays

Science.gov (United States)

Jones, Philip J.; Tomita, Akira; Wartenberg, Mark

1991-08-01

Prototypes of projection displays based on dispersions of liquid crystal in polymer matrices are beginning to appear. The principle of operation depends on electrically switchable light scattering. They are potentially much brighter than current cathode ray tube (CRT) or twisted nematic liquid crystal (TN LC) cell based displays. Comparisons of efficacy and efficiency show this. The contrast and brightness of such displays depend on a combination of the f- number of the projection system and the scattering characteristics of the light valve. Simplified equations can be derived to show these effects. The degree of scattering of current NCAP formulations is sufficient to produce good contrast projection displays, at convenient voltages, that are around three times brighter than TN LC projectors because of the lack of polarizers in the former.

Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

OpenAIRE

Taniguchi, Naohiro; Tokuda, Hajime

2008-01-01

An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

Partial purification of the ATP-driven calcium pump of Streptococcus sanguis

International Nuclear Information System (INIS)

Lynn, A.R.; Rosen, B.P.

1986-01-01

ATP-dependent transport of calcium has been observed in several species of streptococci as uptake of 45 Ca 2+ into everted membrane vesicles. Membranes from Streptococcus sanguis and Streptococcus faecalis were solubilized with octyl-β-D-glucoside or Triton X-100, and the extracts reconstituted into proteoliposomes containing Escherichia coli or soybean phospholipid. Calcium transport in reconstituted proteoliposomes was insensitive to the ionophores nigericin and valinomycin and was unaffected by the F 0 F 1 inhibitor N,N'-dicyclohexylcarbodiimide. Uptake was inhibited by ortho-vanadate with a K/sub i/ in the micromolar range. These results demonstrate that the reconstituted transport activities are not the result of ATP-driven proton pumping via the F 0 F 1 coupled to a calcium/proton antiporter and suggest that existence of a calcium translocating ATPase. Partial purification of the transport activity from Streptococcus sanguis has been achieved using density gradient centrifugation and FPLC

Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

Directory of Open Access Journals (Sweden)

Praveen Balabaskaran Nina

2010-07-01

Full Text Available The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1 sector catalyzes ATP synthesis, whereas the F(o sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1 and F(o sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a

Copper(II) ions interactions in the systems with triamines and ATP. Potentiometric and spectroscopic studies.

Science.gov (United States)

Hoffmann, S K; Goslar, J; Bregier-Jarzebowska, R; Gasowska, A; Zalewska, A; Lomozik, L

2017-12-01

The mode of interaction and thermodynamic stability of complexes formed in binary and ternary Cu(II)/ATP/triamines systems were studied using potentiometric and spectroscopic (NMR, EPR, UV-Vis) methods. It was found that in binary metal-free systems ATP/H x PA species are formed (PA: Spd=spermidine or 3,3-tri=1,7-diamino-4-azaheptane) where the phosphate groups from nucleotides are preferred negative centers and protonated amine groups of amines are positive centers of reaction. In the ternary systems Cu/ATP/H x (PA) as well as Cu/(ATP)(PA) species are formed. The type of the formed Cu(II) complexes depends on pH of the solution. For a low pH value the complexation appears between Cu(II) and ATP molecules via oxygen atoms of phosphate groups. For a very high pH value, where ATP is hydrolyzed, the Cu(II) ions are bound to the nitrogen atoms of polyamine molecules. We did not detect any direct coordination of the N7 nitrogen atom of adenosine to Cu(II) ions. It means that the CuN7 interaction is an indirect type and can be due to noncovalent interplay including water molecule. EPR studies were performed at glassy state (77K) after a fast freezing both for binary and ternary systems. The glassy state EPR spectra do not reflect species identified in titration studies indicating significant effect of rapid temperature decrease on equilibrium of Cu(II) complexes. We propose the molecular structure of all the studied complexes at the glassy state deduced from EPR and optical spectroscopy results. Copyright © 2017 Elsevier Inc. All rights reserved.

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

Science.gov (United States)

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

Relationship between cavitation and loss of echogenicity from ultrasound contrast agents

International Nuclear Information System (INIS)

Radhakrishnan, Kirthi; Kopechek, Jonathan A; Raymond, Jason L; Bader, Kenneth B; Haworth, Kevin J; Holland, Christy K; Huang, Shao-Ling; McPherson, David D

2013-01-01

Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pulse-duration-dependent pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation. Previous studies have demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds and loss of echogenicity of UCAs as a function of pulse duration. Determining the relationship between cavitation thresholds and loss of echogenicity of UCAs would enable monitoring of cavitation based upon the onscreen echogenicity in clinical applications. Two lipid-shelled UCAs, echogenic liposomes (ELIP) and Definity®, were insonified by a clinical ultrasound scanner in duplex spectral Doppler mode at four pulse durations (‘sample volumes’) in both a static system and a flow system. Cavitation emissions from the UCAs insonified by Doppler pulses were recorded using a passive cavitation detection system and stable and inertial cavitation thresholds ascertained. Loss of echogenicity from ELIP and Definity® was assessed within regions of interest on B-mode images. A numerical model based on UCA rupture predicted the functional form of the loss of echogenicity from ELIP and Definity®. Stable and inertial cavitation thresholds were found to have a weak dependence on pulse duration. Stable cavitation thresholds were lower than inertial cavitation thresholds. The power of cavitation emissions was an exponential function of the loss of echogenicity over the investigated range of acoustic pressures. Both ELIP and Definity® lost more than 80% echogenicity before the onset of stable or inertial cavitation. Once this level of echogenicity loss occurred, both stable and inertial cavitation were detected in the physiologic flow phantom. These results imply that stable and

Relationship between cavitation and loss of echogenicity from ultrasound contrast agents.

Science.gov (United States)

Radhakrishnan, Kirthi; Bader, Kenneth B; Haworth, Kevin J; Kopechek, Jonathan A; Raymond, Jason L; Huang, Shao-Ling; McPherson, David D; Holland, Christy K

2013-09-21

Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pulse-duration-dependent pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation. Previous studies have demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds and loss of echogenicity of UCAs as a function of pulse duration. Determining the relationship between cavitation thresholds and loss of echogenicity of UCAs would enable monitoring of cavitation based upon the onscreen echogenicity in clinical applications. Two lipid-shelled UCAs, echogenic liposomes (ELIP) and Definity®, were insonified by a clinical ultrasound scanner in duplex spectral Doppler mode at four pulse durations ('sample volumes') in both a static system and a flow system. Cavitation emissions from the UCAs insonified by Doppler pulses were recorded using a passive cavitation detection system and stable and inertial cavitation thresholds ascertained. Loss of echogenicity from ELIP and Definity® was assessed within regions of interest on B-mode images. A numerical model based on UCA rupture predicted the functional form of the loss of echogenicity from ELIP and Definity®. Stable and inertial cavitation thresholds were found to have a weak dependence on pulse duration. Stable cavitation thresholds were lower than inertial cavitation thresholds. The power of cavitation emissions was an exponential function of the loss of echogenicity over the investigated range of acoustic pressures. Both ELIP and Definity® lost more than 80% echogenicity before the onset of stable or inertial cavitation. Once this level of echogenicity loss occurred, both stable and inertial cavitation were detected in the physiologic flow phantom. These results imply that stable and inertial

Relationship between cavitation and loss of echogenicity from ultrasound contrast agents

Science.gov (United States)

Radhakrishnan, Kirthi; Bader, Kenneth B.; Haworth, Kevin J.; Kopechek, Jonathan A.; Raymond, Jason L.; Huang, Shao-Ling; McPherson, David D.; Holland, Christy K.

2013-09-01

Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pulse-duration-dependent pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation. Previous studies have demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds and loss of echogenicity of UCAs as a function of pulse duration. Determining the relationship between cavitation thresholds and loss of echogenicity of UCAs would enable monitoring of cavitation based upon the onscreen echogenicity in clinical applications. Two lipid-shelled UCAs, echogenic liposomes (ELIP) and Definity®, were insonified by a clinical ultrasound scanner in duplex spectral Doppler mode at four pulse durations (‘sample volumes’) in both a static system and a flow system. Cavitation emissions from the UCAs insonified by Doppler pulses were recorded using a passive cavitation detection system and stable and inertial cavitation thresholds ascertained. Loss of echogenicity from ELIP and Definity® was assessed within regions of interest on B-mode images. A numerical model based on UCA rupture predicted the functional form of the loss of echogenicity from ELIP and Definity®. Stable and inertial cavitation thresholds were found to have a weak dependence on pulse duration. Stable cavitation thresholds were lower than inertial cavitation thresholds. The power of cavitation emissions was an exponential function of the loss of echogenicity over the investigated range of acoustic pressures. Both ELIP and Definity® lost more than 80% echogenicity before the onset of stable or inertial cavitation. Once this level of echogenicity loss occurred, both stable and inertial cavitation were detected in the physiologic flow phantom. These results imply that stable and

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

Science.gov (United States)

Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

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

Science.gov (United States)

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

2010-12-01

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

Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP.

Science.gov (United States)

Sander, Philip; Mostafa, Haouraa; Soboh, Ayman; Schneider, Julian M; Pala, Andrej; Baron, Ann-Kathrin; Moepps, Barbara; Wirtz, C Rainer; Georgieff, Michael; Schneider, Marion

2017-05-23

Glioblastomas (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutic options are urgently needed. A novel drug, Vacquinol-1 (Vac), a quinolone derivative, displays promising properties by inducing rapid cell death in GBM but not in non-transformed tissues. Features of this type of cell death are compatible with a process termed methuosis. Here we tested Vac on a highly malignant glioma cell line observed by long-term video microscopy. Human dental-pulp stem cells (DPSCs) served as controls. A major finding was that an exogenous ATP concentration of as little as 1 μM counter regulated the Vac-induced cell death. Studies using carvacrol, an inhibitor of transient receptor potential cation channel, subfamily M, member 7 (TRPM7), demonstrated that the ATP-inducible inhibitory effect is likely to be via TRPM7. Exogenous ATP is of relevance in GBM with large necrotic areas. Our results support the use of GBM cultures with different grades of malignancy to address their sensitivity to methuosis. The video-microscopy approach presented here allows decoding of signaling pathways as well as mechanisms of chemotherapeutic resistance by long-term observation. Before implementing Vac as a novel therapeutic drug in GBM, cells from each individual patient need to be assessed for their ATP sensitivity. In summary, the current investigation supports the concept of methuosis, described as non-apoptotic cell death and a promising approach for GBM treatment. Tissue-resident ATP/necrosis may interfere with this cell-death pathway but can be overcome by a natural compound, carvacrol that even penetrates the blood-brain barrier.

Determination of ATP as a fluorescence probe with europium(III)-doxycycline.

Science.gov (United States)

Hou, Faju; Wang, Xiaolei; Jiang, Chongqiu

2005-03-01

A new spectrofluorimetric method has been developed for the determination of adenosine disodium triphosphate (ATP). We studied the interactions between the doxycycline (DC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-visible absorption and fluorescence spectra. Using doxycycline (DC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP could remarkably enhance the fluorescence intensity of the DC-Eu3+ complex at lambda = 612 nm. The enhanced fluorescence intensity of the Eu3+ ion was in proportion to the concentration of ATP. The optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP were 1.00 x 10(-7) - 2.00 x 10(-6) mol L(-1) with detection limits of 4.07 x 10(-8) mol L(-1). This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to the determination of ATP in samples. The mechanism of fluorescence enhancement between the doxycycline (DC)-Eu3+ complex and ATP was also studied.

Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus).

Science.gov (United States)

de Kloet, S R

2001-08-01

This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study.

Excessive Extracellular ATP Desensitizes P2Y2 and P2X4 ATP Receptors Provoking Surfactant Impairment Ending in Ventilation-Induced Lung Injury

Directory of Open Access Journals (Sweden)

Djo Hasan

2018-04-01

Full Text Available Stretching the alveolar epithelial type I (AT I cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP (purinergic signaling. Extracellular ATP is cleared by extracellular ATPases, maintaining its homeostasis and enabling the lung to adapt the exocytosis of surfactant to the demand. Vigorous deformation of the AT I cells by high mechanical power ventilation causes a massive release of extracellular ATP beyond the clearance capacity of the extracellular ATPases. When extracellular ATP reaches levels >100 μM, the ATP receptors of the AT II cells become desensitized and surfactant impairment is initiated. The resulting alteration in viscoelastic properties and in alveolar opening and collapse time-constants leads to alveolar collapse and the redistribution of inspired air from the alveoli to the alveolar ducts, which become pathologically dilated. The collapsed alveoli connected to these dilated alveolar ducts are subject to a massive strain, exacerbating the ATP release. After reaching concentrations >300 μM extracellular ATP acts as a danger-associated molecular pattern, causing capillary leakage, alveolar space edema, and further deactivation of surfactant by serum proteins. Decreasing the tidal volume to 6 mL/kg or less at this stage cannot prevent further lung injury.

Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition

Science.gov (United States)

Park, Dohyun; Jeong, Heeyoon; Lee, Mi Nam; Koh, Ara; Kwon, Ohman; Yang, Yong Ryoul; Noh, Jungeun; Suh, Pann-Ghill; Park, Hwangseo; Ryu, Sung Ho

2016-01-01

Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction. PMID:26902888

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

compared to lean control. The ATP synthesis rate without ATP consumption was not different between groups and there were no significant gender differences. The mitochondrial dysfunction in type 2 diabetes in vivo is partly based on a primarily impaired ATP synthesis....... 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...... 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...

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway.

Science.gov (United States)

Montaño, Luis M; Carbajal, Verónica; Vargas, Mario H; García-Hernández, Luz M; Díaz-Hernández, Verónica; Checa, Marco; Barajas-López, Carlos

2013-08-01

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.

Rescue of mitochondrial function in parkin-mutant fibroblasts using drug loaded PMPC-PDPA polymersomes and tubular polymersomes.

Science.gov (United States)

Yealland, G; Battaglia, G; Bandmann, O; Mortiboys, H

2016-09-06

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Effects of Irradiation on bacterial atp luminous intensity of cooled pork and chicken

International Nuclear Information System (INIS)

Ju Hua

2010-01-01

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)

Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

International Nuclear Information System (INIS)

Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

2008-01-01

The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution. Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6 4 22, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å 3 Da −1 , which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan

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.

Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

2014-10-10

Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes.

Science.gov (United States)

Girotti, S; Ferri, E; Cascione, M L; Comuzio, S; Mazzuca, A; Orlandini, A; Breccia, A

1989-07-01

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.

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...... 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 contraction, subjects dropping in force showed lower ATP economy compared with those maintaining the force (3.......7 +/- 0.6 vs 5.3 +/- 0.6 N.s.micromol-1; P contraction could be due to an increase in the ATP economy of contracting muscle fibers offsetting the effects of increased temperature and low ATP economy...

Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

Science.gov (United States)

Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

2018-02-01

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

Directory of Open Access Journals (Sweden)

Milos B. Rokic

2018-04-01

Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

ATP stimulates calcium influx in primary astrocyte cultures

International Nuclear Information System (INIS)

Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

1988-01-01

The effect of ATP and other purines on 45 Ca 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 45 Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45 Ca 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 45 Ca 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 45 Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

Secondary production of the fiddler crab Uca rapax from mangrove areas under anthropogenic eutrophication in the Western Atlantic, Brazil.

Science.gov (United States)

Costa, Tarso de M M; Soares-Gomes, Abilio

2015-12-30

Fiddler crabs Uca rapax were analyzed in three mangrove areas located in both a lagoon and estuarine system in order to study the influence of eutrophication on their population dynamics and production. Populations at the three sites showed a biased sex ratio. Densities were similar at the three sites, but biomass was higher at the lagoon system. Despite biomass being higher at the most eutrophic site, this site exhibited the lowest production. Regarding age structure, the population inhabiting the less eutrophic site mainly comprised younger crabs. The lower production and smaller P/B ratio found in the more eutrophic site were most likely consequences of a high mortality rate and an aged population. Our study evidences the high plasticity of the fiddler crab U. rapax, and confirms secondary production and P/B ratio estimates as useful tools to assess the effects of environmental change. Copyright © 2015 Elsevier Ltd. All rights reserved.

CFTR mediates noradrenaline-induced ATP efflux from DRG neurons.

Science.gov (United States)

Kanno, Takeshi; Nishizaki, Tomoyuki

2011-09-24

In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β(3) adrenoceptors linked to G(s) protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel.

Small amounts of functional ATP7A protein permit mild phenotype

DEFF Research Database (Denmark)

Møller, Lisbeth Birk

2015-01-01

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...... 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...

microRNA dependent and independent deregulation of long non-coding RNAs by an oncogenic herpesvirus.

Directory of Open Access Journals (Sweden)

Sunantha Sethuraman

2017-07-01

Full Text Available Kaposi's sarcoma (KS is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi's sarcoma-associated herpesvirus (KSHV is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL. In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs. The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.

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, ou...... may be important in pancreas physiology and potentially in pancreas pathophysiology....... 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...

ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

Directory of Open Access Journals (Sweden)

Romina Baaske

2016-12-01

Full Text Available Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla. This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L, which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin.

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1 adenosinetriphosphatase in the presence of dimethyl sulfoxide

International Nuclear Information System (INIS)

Kandpal, R.P.; Stempel, K.E.; Boyer, P.D.

1987-01-01

Addition of dimethyl sulfoxide promotes the formation of enzyme-bound ATP from medium P/sub i/ by mitochondrial F 1 adenosinetriphosphatase that has tightly bound ADP present. Measurements are reported of medium P/sub i/ ↔ H 18 OH exchange and of the dependence of formation of enzyme-bound ATP on P/sub i/ concentration. Attainment of an apparent equilibrium between medium P/sub i/ and bound ATP requires longer than 30 min, even though the rates of P/sub i/ binding and release after apparent equilibrium is reached would suffice for a faster approach to equilibrium. Slow protein conformational changes or other unknown modulating factors may be responsible for the slow rate of bound ATP formation. After apparent equilibrium is reached, each P/sub i/ that binds to the enzyme reversibly forms ATP about 50 times before being released to the medium. The rate of interconversion of bound ATP to bound ADP and P/sub i/ is much slower than that in the absence of dimethyl sulfoxide as measured with sufficient low ATP concentrations so that single-site catalysis is favored. Although the interconversion rate is slowed, the equilibrium constant for bound ATP formation from bound ADP and P/sub i/ is not far from unity. Dimethyl sulfoxide favors the formation of enzyme-bound ATP by promoting the competent binding of P/sub i/ to enzyme with ADP bound at a catalytic site rather than by promoting formation of bound ATP from bound ADP and P/sub i/

Synthesis and purification of [γP32]-ATP

International Nuclear Information System (INIS)

Kukuh, Ratnawati; Santoso, Daniel; Basri, T. Hasan; Natalia Adventini

1995-01-01

The synthesis of [γP 3 2]-ATP has been carried out using an enzymes procedure. The compound was formed by the phosphorylation of ADP during the enzymatic conversion of L-α-glycerol-phosphate to 3-phosphoglycerate. In the present study, lactatedehydrogenase and sodium pyruvat were used in order to maintain β-NAD + concentration and to push the reaction of glyceralaldehyde-3-phosphate dehydrogenase towards the formation of 1,3-diphosphoglycerate. L-α-glycerolphosphate was used as primary substrate, as it is more stable than DL-glyceraldehyde-3-phosphate. The enzymatic reaction was stopped by immersing the reaction vessel in boiling water for about 10 minutes. The labelled [γP 3 2]-ATP formed was separated by thin layer chromatography using PEI-cellulose and the spots of [γP 3 2]-ATP and inorganic P 3 2 residue located by autoradiography using X-ray film. The optimum time for the reaction at room temperature was 90 minutes with a labeling efficiency of 94.9 %. Purification of the [γP 3 2]-ATP by anion exchange chromatography using DEAE sephadex yielded a purity of more than 95%. The results showed that the labeled compound [γP 3 2]-ATP can be synthesized via an enzymatic process with a satisfactory yield. (author), 4 refs, 2 tabs, 2 figs

New control method of on-board ATP system of Shinkansen trains

Energy Technology Data Exchange (ETDEWEB)

Fukuda, N.; Watanabe, T. [Railway Technical Research Inst. (Japan)

2000-07-01

We studied a new control method of the on-board automatic train protection (ATP) system for Shinkansen trains to shorten the operation time and not to degrade ride comfort at changes in deceleration of the train, while maintaining the safety and reliability of the present ATP signal system. We propose a new on-board pattern brake control system based on the present ATP data without changing the wayside equipment. By simulating the ATP braking of the proposed control method, we succeeded in shortening the operation time by 48 seconds per one station in comparison with the present ATP brake control system. This paper reports the concept of the system and simulation results of the on-board pattern. (orig.)

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

Science.gov (United States)

Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

2015-09-22

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Application of luciferase assay for ATP to antimicrobial drug susceptibility

Science.gov (United States)

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

1977-01-01

The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

Maitotoxin-induced liver cell death involving loss of cell ATP following influx of calcium

International Nuclear Information System (INIS)

Kutty, R.K.; Singh, Y.; Santostasi, G.; Krishna, G.

1989-01-01

Maitotoxin, one of the most potent marine toxins known, produced cell death in cultures of rat hepatocytes with a TD50 of 80 pM at 24 hr. The cell death, as indicated by a dose- and time-dependent leakage of lactate dehydrogenase (LDH), was also associated with the leakage of [14C]adenine nucleotides from hepatocytes prelabeled with [14C]-adenine. The toxic effect of maitotoxin was completely abolished by the omission of calcium from the culture medium. The cell death induced by maitotoxin increased with increasing concentrations of calcium in the medium. Treatment of hepatocytes with low concentrations of the toxin (less than 0.5 ng/ml) resulted in increases in 45Ca influx into the cells. At higher concentrations of maitotoxin (greater than 1ng/ml), the initial increase in 45Ca influx was followed by the release of the 45Ca from the cells into the medium. Since the 45Ca release paralleled the LDH leakage, the release of calcium was due to cell death. The 45Ca influx, [14C]adenine nucleotide leakage, and LDH leakage were effectively inhibited by verapamil, a calcium channel blocker. Maitotoxin also induced a time- and dose-dependent loss of ATP from hepatocytes, which preceded the [14C]adenine nucleotide and LDH leakage. Thus, it appears that the cell death resulting from maitotoxin treatment is caused by the elevated intracellular calcium, which in turn inhibits mitochondrial oxidative phosphorylation causing depletion of cell ATP. Loss of cell ATP may be the causative event in the maitotoxin-induced cell death

atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

Science.gov (United States)

2013-01-01

Background The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. Results Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. Conclusions The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples. PMID:24299240

Sarcolemmal cardiac K(ATP) channels as a target for the cardioprotective effects of the fluorine-containing pinacidil analogue, flocalin.

Science.gov (United States)

Voitychuk, Oleg I; Strutynskyi, Ruslan B; Yagupolskii, Lev M; Tinker, Andrew; Moibenko, Olexiy O; Shuba, Yaroslav M

2011-02-01

A class of drugs known as K(ATP) -channel openers induce cardioprotection. This study examined the effects of the novel K(ATP) -channel opener, the fluorine-containing pinacidil derivative, flocalin, on cardiac-specific K(ATP) -channels, excitability of native cardiac myocytes and on the ischaemic heart. The action of flocalin was investigated on: (i) membrane currents through cardiac-specific K(ATP) -channels (I(KATP) ) formed by K(IR) 6.2/SUR2A heterologously expressed in HEK-293 cells (HEK-293(₆.₂/₂A) ); (ii) excitability and intracellular Ca²(+) ([Ca²(+) ](i) ) transients of cultured rat neonatal cardiac myocytes; and (iii) functional and ultrastructural characteristics of isolated guinea-pig hearts subjected to ischaemia-reperfusion. Flocalin concentration-dependently activated a glibenclamide-sensitive I(KATP) in HEK-293(₆.₂/₂A) cells with an EC₅₀= 8.1 ± 0.4 µM. In cardiac myocytes, flocalin (5 µM) hyperpolarized resting potential by 3-5 mV, markedly shortened action potential duration, reduced the amplitude of [Ca²(+) ](i) transients by 2-3-fold and suppressed contraction. The magnitude and extent of reversibility of these effects depended on the type of cardiac myocytes. In isolated hearts, perfusion with 5 µmol·L⁻¹ flocalin, before inducing ischaemia, facilitated restoration of contraction during reperfusion, decreased the number of extrasystoles, prevented the appearance of coronary vasoconstriction and reduced damage to the cardiac tissue at the ultrastructural level (state of myofibrils, membrane integrity, mitochondrial cristae structure). Flocalin induced potent cardioprotection by activating cardiac-type K(ATP) -channels with all the benefits of the presence of fluorine group in the drug structure: higher lipophilicity, decreased toxicity, resistance to oxidation and thermal degradation, decreased metabolism in the organism and prolonged therapeutic action. © 2011 The Authors. British Journal of Pharmacology © 2011 The

ATP Synthase, a Target for Dementia and Aging?

Science.gov (United States)

Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

2018-02-01

Advancing age is the biggest risk factor for development for the major life-threatening diseases in industrialized nations accounting for >90% of deaths. Alzheimer's dementia (AD) is among the most devastating. Currently approved therapies fail to slow progression of the disease, providing only modest improvements in memory. Recently reported work describes mechanistic studies of J147, a promising therapeutic molecule previously shown to rescue the severe cognitive deficits exhibited by aged, transgenic AD mice. Apparently, J147 targets the mitochondrial alpha-F1-ATP synthase (ATP5A). Modest inhibition of the ATP synthase modulates intracellular calcium to activate AMP-activated protein kinase to inhibit mammalian target of rapamycin, a known mechanism of lifespan extension from worms to mammals.

Assembly of the membrane domain of ATP synthase in human mitochondria.

Science.gov (United States)

He, Jiuya; Ford, Holly C; Carroll, Joe; Douglas, Corsten; Gonzales, Evvia; Ding, Shujing; Fearnley, Ian M; Walker, John E

2018-03-20

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

Induction of suppression of delayed type hypersensitivity to herpes simplex virus by epidermal cells exposed to UV-irradiated urocanic acid in vivo

International Nuclear Information System (INIS)

Ross, J.A.; Howie, S.E.; Norval, M.; Maingay, J.P.

1987-01-01

Urocanic acid (UCA), the putative photoreceptor for ultraviolet radiation (UV)-induced suppression, undergoes a UV-dependent trans to cis isomerisation. Epidermal cells from mice painted with UCA, containing a known proportion of the cis-isomer, generate suppression of the delayed type hypersensitivity response to herpes simplex virus type 1 (HSV-1) when transferred to naive syngeneic recipients at the same time and site as infection with HSV-1. One T suppressor cell subset, of phenotype (Thy1+, L3T4+, Ly2-), is induced by the cis-UCA modified epidermal cell transfer. Flow cytometric analysis of the epidermal cells from skin treated with UV or cis-UCA indicates an overall reduction from normal in the number of cells expressing MHC Class II antigens, but no alteration in the number expressing I-J antigens

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.

Target-assistant Zn2+-dependent DNAzyme for signal-on electrochemiluminescent biosensing

International Nuclear Information System (INIS)

Huang, Yin; Lei, Jianping; Cheng, Yan; Ju, Huangxian

2015-01-01

Highlights: • The sensing strategy is based on cleavage reaction of target-assistant Zn 2+ -dependent DNAzyme. • A dual quenching mechanism of ECL is identified. • A sensitive and selective ECL sensor is constructed for detection of ATP. • The biosensor can detect ATP in serum samples with good accuracy. - Abstract: A signal-on electrochemiluminescent (ECL) approach for ultrasensitive ATP detection was developed using target-assistant Zn 2+ -dependent DNAzyme via a dual quenching pathway between quantum dots (QDs) and Au nanoclusters (Au NCs). The facile ECL biosensor was constructed by covalent assembly of Au NCs-labeled hairpin DNA on QDs modified glassy carbon electrode. A dual quenching ECL mechanism was identified to be via resonance energy transfer between QDs and Au NCs and electrocatalytic reduction of coreactant oxygen by Au NCs. With the assistance of two help DNAs, the G-quadruplex structure of ATP aptamer was formed, and thus narrowed the two fragments of Zn 2+ -dependent DNAzyme. In the presence of Zn 2+ , Zn 2+ -dependent DNAzyme can be generated in situ on the biosensor's surface. The as-prepared DNAzyme can cleave the substrate strand, and release the Au NCs from the electrode, resulting in the signal-on ECL state. This biosensor showed good analytical performance with 4 orders magnitude linear range, excellent specificity, and acceptable stability. The biosensor had been applied in detection of ATP in real serum sample and provided significant potential application in clinical analysis

Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

Science.gov (United States)

Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

2015-03-01

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

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 - others: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

Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.

LENUS (Irish Health Repository)

Pitceathly, Robert D S

2012-09-11

Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

Selective geochemistry of iron in mangrove soils in a semiarid tropical climate: effects of the burrowing activity of the crabs Ucides cordatus and Uca maracoani

Science.gov (United States)

Araújo, J. M. C.; Otero, X. L.; Marques, A. G. B.; Nóbrega, G. N.; Silva, J. R. F.; Ferreira, T. O.

2012-08-01

Bioturbation by crabs may affect processes associated with organic matter decomposition in mangrove soils. This study examines how two crabs ( Uca maracoani and Ucides cordatus), which are of substantial ecological and economic importance in semiarid coastal areas of Brazil, affect biogeochemical processes in mangrove soils. For this purpose, the physicochemical and geochemical parameters of the soils at different sites were analyzed. The redox potential was always positive at bioturbated sites (+12 to +218 mV), indicating more oxidizing conditions conducive to the oxidation of pyrite and precipitation of oxyhydroxides. In contrast, anoxic conditions prevailed at the control site (Eh mangrove soils, being capable of enhancing organic matter decomposition and also shifting the dominant pathway of organic matter degradation.

Processing mechanics of alternate twist ply (ATP) yarn technology

Science.gov (United States)

Elkhamy, Donia Said

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

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

Directory of Open Access Journals (Sweden)

Hao Ding

Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

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

Science.gov (United States)

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

2016-03-15

Fundamental criticisms have been made over the use of (31)P 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 (31)P 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. Copyright © 2016 the American Physiological Society.

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

Science.gov (United States)

Park, Yoojin; Nim-Anussornkul, Duangrat; Vilaivan, Tirayut; Morii, Takashi; Kim, Byeang Hyean

2018-01-15

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct measurement of newly synthesized ATP dissociation kinetics in sarcoplasmic reticulum ATPase

International Nuclear Information System (INIS)

Teruel-Puche, J.; Kurzmack, M.; Inesi, G.

1987-01-01

Incubation of SR vesicles with Ca 2+ and ( 32 P)acetylphosphate, yields steady state levels of ( 32 P)phosphorylated enzyme (ATPase) intermediate and high concentrations of Ca 2+ in the lumen of the vesicles. At this time, addition of ADP (and EGTA to lower the Ca 2+ concentration in the medium outside the vesicles) results in single cycle formation of (γ- 32 P)ATP by transfer of ( 32 P)phosphate from the enzyme intermediate to ADP. The phosphoenzyme decay and ATP formation exhibit a fast component within the first 20 msec following addition of ADP, and a slower component reaching an asymptote in approximately 100 msec. They have now measured by a rapid filtration method the fraction of newly synthesized ATP which is bound to the enzyme, as opposed to the fraction dissociated into the medium. They find that nearly all the ATP formed during the initial burst is still bound to the enzyme within the initial 20 msec of reaction. Dissociation of newly synthesized ATP occurs then with approximately 13 sec -1 rate constant, permitting reequilibration of the system and further formation of ATP. The rate limiting effect of ATP dissociation and other partial reactions on the slow component of single cycle ATP synthesis is evaluated by appropriate kinetic simulations

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

Science.gov (United States)

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Antihypertrophic Effects of Small Molecules that Maintain Mitochondrial ATP Levels Under Hypoxia

Directory of Open Access Journals (Sweden)

Hiroaki Nagai

2017-10-01

Full Text Available Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin β, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.

ATP regeneration with enzymes of the alcohol fermentation pathway and kinases of yeast and its computer simulation

Energy Technology Data Exchange (ETDEWEB)

Asada, M; Shirai, Y; Nakanishi, K; Matsuno, R; Kimura, A; Kamikubo, T

1981-08-01

Enzymes of the alcohol fermentation pathway, adenosine kinase and adenylate kinase were extracted by incubating acetone-dried yeast cells with a simple reaction medium containing glucose, and subjected to gel filtration to remove the intermediates of alcohol fermentation formed during incubation. By using the enzyme systems as catalysts and glucose as an energy source, ATP was regenerated from adenosine. A minimum concentration of fructose 1,6-bisphosphate (FBP) of 7 mM or of ATP of 10 mM was necessary to initiate the alcohol fermentation; and concentrations of nucleotides changed abruptly with reaction time. These nonlinear phenomena might be due to action of FBPase and/or ATPase as well as the complex multienzyme systems. To understand the experimentally observed phenomena, a mathematical model of the reaction system was proposed which takes into account ATP regeneration. The calculated time-dependent concentrations of glucose, FBP, adenosine and nucleotides were in agreement with experimental values qualitatively as well as quantitatively. Moreover, the nonlinear phenomena, that is, the existence of threshold concentrations of FBP and ATP below which the reaction can not proceed and the steep changes of nucleotide concentrations were also accounted for. These results indicate that the model was quite suitable for this reaction system and useful for predicting the experimental results.

A comparative study of ATPase subunit 9 (Atp9) gene between ...

African Journals Online (AJOL)

ATPase subunit 9 gene (Atp9) is an important functional gene in mitochondria, and is closely related with energy supply. RNA editing of atp9 gene was associated with male sterility in plants. In this study, the atp9 gene in soybeans was cloned from a soybean cytoplasmic male sterile line NJCMS2A and its maintainer line ...

Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients

Science.gov (United States)

Lahner, Edith; Brigatti, Cristina; Marzinotto, Ilaria; Carabotti, Marilia; Scalese, Giulia; Davidson, Howard W; Wenzlau, Janet M; Bosi, Emanuele; Piemonti, Lorenzo; Annibale, Bruno; Lampasona, Vito

2017-01-01

Objectives: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls. Methods: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies. Results: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (Pgastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa. PMID:28102858

Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

Directory of Open Access Journals (Sweden)

Pengchong Jiang

Full Text Available Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme, suramin (broad-spectrum P2 receptor antagonist, 2-APB (IP3 receptor blocker and thapsigargin (endoplasmic reticulum calcium pump inhibitor potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

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

Science.gov (United States)

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

2013-01-01

We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

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 ...

Engineering an ATP-dependent D-Ala:D-Ala ligase for synthesizing amino acid amides from amino acids.

Science.gov (United States)

Miki, Yuta; Okazaki, Seiji; Asano, Yasuhisa

2017-05-01

We successfully engineered a new enzyme that catalyzes the formation of D-Ala amide (D-AlaNH 2 ) from D-Ala by modifying ATP-dependent D-Ala:D-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of D-Ala-D-Ala from two molecules of D-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second D-Ala of D-Ala-D-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for D-AlaNH 2 production. The S293E variant, which was selected as the best enzyme for D-AlaNH 2 production, exhibited an optimal activity at pH 9.0 and 40 °C for D-AlaNH 2 production. The apparent K m values of this variant for D-Ala and NH 3 were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of D-AlaNH 2 from 10 and 50 mM D-Ala and 3 M NH 4 Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.

Possible involvement of ATP-sensitive potassium channels in the antidepressant-like effect of baclofen in mouse forced swimming test.

Science.gov (United States)

Nazari, Seyedeh Khadijeh; Nikoui, Vahid; Ostadhadi, Sattar; Chegini, Zahra Hadi; Oryan, Shahrbanoo; Bakhtiarian, Azam

2016-12-01

Previous study confirmed that the acute treatment with baclofen by inhibition of the l-arginine-nitric oxide (NO) pathway diminished the immobility behavior in the forced swimming test (FST) of mice. Considering the involvement of NO in adenosine triphosphate (ATP)-sensitive potassium channels (K ATP ), in the present study we investigated the involvement of K ATP channels in antidepressant-like effect of baclofen in the forced swimming test (FST). After assessment of locomotor behavior in the open-field test (OFT), FST was applied for evaluation of the antidepressant-like activity of baclofen in mice. Baclofen at different doses (0.1, 0.3, and 1mg/kg) and fluoxetine (20mg/kg) were administrated by intraperitoneal (ip) route, 30min before the FST or OFT. To clarify the probable involvement of K ATP channels, after determination of sub-effective doses of glibenclamide as a K ATP channel blocker and cromakalim, as an opener of these channels, they were co-administrated with the sub-effective and effective doses of baclofen, respectively. Baclofen at dose 1mg/kg significantly decreased the immobility behavior of mice similar to fluoxetine (20mg/kg). Co-administration of gelibenclamide sub-effective dose (1mg/kg) with baclofen (0.1mg/kg) showed a synergistic antidepressant-like effect in the FST. Also, sub-effective dose of cromakalim (0.1mg/kg) inhibited the antidepressant-like effect of baclofen (1mg/kg) in the FST. All aforementioned treatments had not any impact on the locomotor movement of mice in OFT. Our study for the first time revealed that antidepressant-like effect of baclofen on mice is K ATP -dependent, and baclofen seems that exert this effect by blocking the K ATP channels. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Excessive extracellular ATP desensitizes P2Y2 and P2X4 ATP receptors provoking surfactant impairment ending in ventilation-induced lung injury

NARCIS (Netherlands)

D. Hasan (Djo); Satalin, J. (Joshua); van der Zee, P. (Philip); Kollisch-Singule, M. (Michaela); P. Blankman (Paul); Shono, A. (Atsuko); P. Somhorst (Peter); C.A. den Uil (Corstiaan); H.J. Meeder (Han); Kotani, T. (Toru); G.F. Nieman (Gary F.)

2018-01-01

textabstractStretching the alveolar epithelial type I (AT I) cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP) (purinergic signaling). Extracellular ATP is cleared by extracellular ATPases,

Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

Science.gov (United States)

Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

2013-07-15

ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

Spectrographic study of neodymium complexing with ATP and ADP

International Nuclear Information System (INIS)

Svetlova, I.E.; Dobrynina, N.A.; Martynenko, L.N.

1989-01-01

By spectrographic method neodymium complexing with ATP and ADP in aqueous solutions at different pH values has been studied. The composition of the complexes was determined by the method of isomolar series. On the basis of analysis of absorption spectra it has been ascertained that at equimolar ratio of Nd 3+ and ATP absorption band of L278A corresponds to monocomplex, and the band of 4290 A - to biscomplex. For the complexes with ADP the absorption band of 4288 A is referred to bicomplexes. The character of ATP and ADP coordination by Nd 3+ ion is considered. Stability constants of the complexes are calculated

ATP synthase--a marvellous rotary engine of the cell.

Science.gov (United States)

Yoshida, M; Muneyuki, E; Hisabori, T

2001-09-01

ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

Science.gov (United States)

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

[Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

Science.gov (United States)

Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

2015-08-01

Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals.

The P2X7 ATP receptor modulates renal cyst development in vitro

International Nuclear Information System (INIS)

Hillman, Kate A.; Woolf, Adrian S.; Johnson, Tanya M.; Wade, Angela; Unwin, Robert J.; Winyard, Paul J.D.

2004-01-01

P2X 7 , a piercing receptor, is expressed in renal collecting ducts as they undergo fulminant cysto genesis in the cpk/cpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Dissociated cpk/cpk kidneys generate cysts from cell aggregates within 24 h of suspension culture and we demonstrate that BzATP, a P2X 7 agonist, reduces cystogenesis. This effect is P2X 7 -specific, because: (i) equimolar concentrations of other purinergic agonists, ATP and UTP, had lesser effects and (ii) the P2X 7 inhibitor, oxidized ATP, abrogated the BzATP-mediated reduction in cystogenesis. BzATP did not significantly affect total cell number, proliferation, LDH release or caspase 3 activity, and zVAD-fmk, a caspase blocker, failed to modulate BzATP effects. In addition, this P2X 7 agonist did not significantly alter cyst size, probably excluding altered vectorial transport. In vivo, ATP was detected in cyst fluid from cpk/cpk kidneys; moreover, P2X 7 protein was also upregulated in human fetal ARPKD epithelia versus normal fetal collecting ducts. Thus, ATP may inhibit pathological renal cyst growth through P2X 7 signaling

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.

Role of connexin 32 hemichannels in the release of ATP from peripheral nerves.

Science.gov (United States)

Nualart-Marti, Anna; del Molino, Ezequiel Mas; Grandes, Xènia; Bahima, Laia; Martin-Satué, Mireia; Puchal, Rafel; Fasciani, Ilaria; González-Nieto, Daniel; Ziganshin, Bulat; Llobet, Artur; Barrio, Luis C; Solsona, Carles

2013-12-01

Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy. Copyright © 2013 Wiley Periodicals, Inc.

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

DEFF Research Database (Denmark)

Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki

2012-01-01

and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation...... affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3....

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 an ATP13A2 mutation (one affected mutation carrier (MC) with a compound heterozygous mutation, 4 asymptomatic MC with a single heterozygous mutation) and 11 healthy subjects without mutations were studied. We measured motor evoked potentials (MEP), the contralateral silent period (cSP), short interval....... RESULTS: CSP duration was increased in the symptomatic ATP13A2 MC. The iSP measurements revealed increased interhemispheric inhibition in both the compound heterozygous and the heterozygous MC. CONCLUSION: A compound heterozygous mutation in the ATP13A2 gene is associated with increased intracortical...

MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

Science.gov (United States)

Zhao, Yumei; Migita, Keisuke; Sun, Jing; Katsuragi, Takeshi

2010-04-01

Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

Exploration of charge states of balanol analogues acting as ATP-competitive inhibitors in kinases.

Science.gov (United States)

Hardianto, Ari; Yusuf, Muhammad; Liu, Fei; Ranganathan, Shoba

2017-12-28

(-)-Balanol is an ATP mimic that inhibits protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA) with limited selectivity. While PKA is a tumour promoter, PKC isozymes act as tumour promoters or suppressors, depending on the cancer type. In particular, PKCε is frequently implicated in cancer promotion, making it a potential target for anticancer drugs. To improve isozyme selectivity of balanol, exhaustive structural and activity relationship (SAR) studies have been performed in the last two decades, but with limited success. More recently, fluorination on balanol has shown improved selectivity for PKCε, although the fluorine effect is not yet clearly understood. Understanding the origin to this fluorine-based selectivity will be valuable for designing better balanol-based ATP mimicking inhibitors. Computational approaches such as molecular dynamics (MD) simulations can decipher the fluorine effect, provided that correct charges have been assigned to a ligand. Balanol analogues have multiple ionisable functional groups and the effect of fluorine substitutions on the exact charge state of each analogue bound to PKA and to PKCε needs to be thoroughly investigated in order to design highly selective inhibitors for therapeutic applications. We explored the charge states of novel fluorinated balanol analogues using MD simulations. For different potential charge states of these analogues, Molecular Mechanics Generalized Born Surface Area (MMGBSA) binding energy values were computed. This study suggests that balanol and the most potent fluorinated analogue (5S fluorine substitution on the azepane ring), have charges on the azepane ring (N1), and the phenolic (C6''OH) and the carboxylate (C15''O 2 H) groups on the benzophenone moiety, when bound to PKCε as well as PKA. To the best our knowledge, this is the first study showing that the phenolate group is charged in balanol and its analogues binding to the ATP site of PKCε. Correct charge

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

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 measurements for monitoring microbial drinking water quality

DEFF Research Database (Denmark)

Vang, Óluva Karin

Current standard methods for surveillance of microbial drinking water quality are culture based, which are laborious and time-consuming, where results not are available before one to three days after sampling. This means that the water may have been consumed before results on deteriorated water....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP......, microbial quality in distributed water, detection of aftergrowth, biofilm formation etc. This PhD project demonstrated that ATP levels are relatively low and fairly stable in drinking water without chlorine residual despite different sampling locations, different drinking water systems and time of year...

Electrochemical Investigation of the Interaction between Catecholamines and ATP.

Science.gov (United States)

Taleat, Zahra; Estévez-Herrera, Judith; Machado, José D; Dunevall, Johan; Ewing, Andrew G; Borges, Ricardo

2018-02-06

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

Science.gov (United States)

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-06-17

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

Science.gov (United States)

Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

2012-12-01

Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

Science.gov (United States)

MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

2007-06-01

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Silencing of Atp2b1 increases blood pressure through vasoconstriction.

Science.gov (United States)

Shin, Young-Bin; Lim, Ji Eun; Ji, Su-Min; Lee, Hyeon-Ju; Park, So-Yon; Hong, Kyung-Won; Lim, Mihwa; McCarthy, Mark I; Lee, Young-Ho; Oh, Bermseok

2013-08-01

Recent genome-wide association studies (GWASs) have identified 30 genetic loci that regulate blood pressure, increasing our understanding of the cause of hypertension. However, it has been difficult to define the causative genes at these loci due to a lack of functional analyses. In this study, we aimed to validate the candidate gene ATP2B1 in 12q21, variants near which have the strongest association with blood pressure in Asians and Europeans. ATP2B1 functions as a calcium pump to fine-tune calcium concentrations - necessary for repolarization following muscular contractions. We silenced Atp2b1 using an siRNA complex, injected into mouse tail veins. In treated mice, blood pressure rose and the mesenteric arteries increased in wall : lumen ratio. Moreover, the arteries showed enhanced myogenic responses to pressure, and contractile responses to phenylephrine increased compared with the control, suggesting that blood pressure is regulated by ATP2B1 through the contraction and dilation of the vessel, likely by controlling calcium concentrations in the resting state. These results support that ATP2B1 is the causative gene in the blood pressure-associated 12q21 locus and demonstrate that ATP2B1 expression in the vessel influences blood pressure.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

Science.gov (United States)

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.

Science.gov (United States)

Shyng, S L; Barbieri, A; Gumusboga, A; Cukras, C; Pike, L; Davis, J N; Stahl, P D; Nichols, C G

2000-01-18

ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.

Extracellular ATP acts as a damage associated molecular pattern (DAMP signal in plants

Directory of Open Access Journals (Sweden)

Kiwamu eTanaka

2014-09-01

Full Text Available As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs. ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling role in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor Kinase, which is plant-specific. P2K1 (DORN1 is required for ATP-induced cellular responses (e.g., cytosolic Ca2+ elevation, MAPK phosphorylation, and gene expression. Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of the future research of extracellular ATP as a DAMP signal.

Effect of irradiation on detection of bacteria in dehydrated vegetables with ATP bioluminescence assay

International Nuclear Information System (INIS)

Xiao Huan; Luo Shishi; Wang Zegang; Feng Min; Zhu Jiating; Chen Xiulan; Zhai Jianqing

2011-01-01

ATP bioluminescence intensity of 4 kinds of irradiated dehydrated vegetables was inconsistent with the bacteria number, the reasons were investigated in this paper. Results showed that irradiation had little effect on background luminescence, and there was no effect on luciferase-luminous system. When irradiation killed the bacteria, the ATPase activity also decreased. As a result, the ATP content in bacteria didn't decreased with the killed of bacteria, which contributed to the increase of free ATP in ATP extract and finally led to the disagreement between the bioluminescence intensity and the actual number of bacteria. When the free ATP in the dehydrated vegetable was removed, the bioluminescence intensity of ATP extract was consistent with the actual number of bacteria in irradiated dehydrated vegetable and ATP bioluminescence technology could be used in bacteria detection of irradiated samples. (authors)

Tomatidine Is a Lead Antibiotic Molecule That Targets Staphylococcus aureus ATP Synthase Subunit C.

Science.gov (United States)

Lamontagne Boulet, Maxime; Isabelle, Charles; Guay, Isabelle; Brouillette, Eric; Langlois, Jean-Philippe; Jacques, Pierre-Étienne; Rodrigue, Sébastien; Brzezinski, Ryszard; Beauregard, Pascale B; Bouarab, Kamal; Boyapelly, Kumaraswamy; Boudreault, Pierre-Luc; Marsault, Éric; Malouin, François

2018-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro -generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10 5 -fold for FC04-100. Copyright © 2018 American Society for Microbiology.

The lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

Energy Technology Data Exchange (ETDEWEB)

Otoikhian, Adenike [Oregon Health & Sciences University; Barry, Amanda N. [Los Alamos National Laboratory; Mayfield, Mary [Oregon Health & Science University; Nilges, Mark [Illinois EPR Center; Huang, Yiping [Johns Hopkins University; Lutsenko, Svetlana [Johns Hopkins University; Blackburn, Ninian [Oregon Health & Science University

2012-05-14

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

Science.gov (United States)

Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

2013-07-01

A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

Diverse Functional Properties of Wilson Disease ATP7B Variants

Science.gov (United States)

Huster, Dominik; Kühne, Angelika; Bhattacharjee, Ashima; Raines, Lily; Jantsch, Vanessa; Noe, Johannes; Schirrmeister, Wiebke; Sommerer, Ines; Sabri, Osama; Berr, Frieder; Mössner, Joachim; Stieger, Bruno; Caca, Karel; Lutsenko, Svetlana

2012-01-01

BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis. PMID:22240481

ATPS: "Aqueous two-phase System" as the "Answer to Protein Separation" for protein-processing Food Industry.

Science.gov (United States)

Khan, Bilal Muhammad; Liu, Zhi-Cong; Shi, Fu-Lin; Cheong, Kit-Leong; Liu, Yang

2018-06-08

Every individual needs food for its nutritional value and flavor while the economic growth of a nation depends on a thriving profit-generating industry. The food industry caters to both needs in an efficient manner. Proteins can rightly be considered as the driving force behind the overwhelming success of this industry. However, purification of proteins is not an easy undertaking due to their intricate nature while presently employed procedures for this purpose, regrettably, are both costly, and labor- and time-intensive in addition to being unsettling on proteins structural conformity. ATPS has accumulated a lot of interest from the scientific community due to its mild operating conditions, high recovery yield, ease of scaling it up, and its cost-effective and environment friendly nature. This review tries to amass some accounts concerning the utility of ATPS for the separation and purification of proteins. Some auspicious clues in this regard can be witnessed along with a few loopholes which need to be addressed before this technique can truly demonstrate its potential vis-à-vis industrial protein purification. Overall, a polymer - salt (citrates in particular) ATPS with an added inert supplementary salt can be regarded as a better option for purifying proteins.

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

Directory of Open Access Journals (Sweden)

Askew Christopher D

2009-12-01

Full Text Available Abstract Background It has been proposed that adenosine triphosphate (ATP released from red blood cells (RBCs may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC. Results Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P Conclusions Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

The danger signal extracellular ATP is an inducer of Fusobacterium nucleatum biofilm dispersal

Directory of Open Access Journals (Sweden)

Qinfeng Ding

2016-11-01

Full Text Available Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP is an example of a danger associated molecular pattern (DAMP employed by mammalian cells to elicit inflammatory and damage healing responses. Although the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were significantly more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also exhibited significantly higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a

Molecular structure of human KATP in complex with ATP and ADP.

Science.gov (United States)

Lee, Kenneth Pak Kin; Chen, Jue; MacKinnon, Roderick

2017-12-29

In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K + channel Kir6.2, in the presence of Mg 2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg 2+ -ATP in the degenerate site and Mg 2+ -ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP's ability to override ATP inhibition. © 2017, Lee et al.

Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

Directory of Open Access Journals (Sweden)

Tomomi Ando

Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

Children's Control/Display Stereotypes.

Science.gov (United States)

Hoffmann, Errol R; Chan, Alan H S; Tai, Judy P C

2018-06-01

Objective The aim of this study was to determine control/display stereotypes for children of a range of ages and development of these stereotypes with age. Background Little is known about control/display stereotypes for children of different ages and the way in which these stereotypes develop with age. This study is part of a program to determine the need to design differentially for these age groups. Method We tested four groups of children with various tasks (age groups 5 to 7, 8 to 10, 11 to 13, 14 to 16), with about 30 in each group. Examples of common tasks were opening a bottle, turning on taps, and allocating numbers to keypads. More complex tasks involved rotating a control to move a display in a requested direction. Results Tasks with which different age groups were familiar showed no effect of age group. Different control/display arrangements generally showed an increase in stereotype strength with age, with dependence on the form of the control/display arrangement. Two-dimensional arrangements, with the control on the same plane as the display, had higher stereotype strength than three-dimensional arrangements for all age groups, suggesting an effect of familiarity with controls and displays with increasing age. Conclusion Children's control/display stereotypes do not differ greatly from those of adults, and hence, design for children older than 5 years of age, for control/display stereotypes, can be the same as that for adult populations. Application When designing devices for children, the relationship between controls and displays can be as for adult populations, for which there are considerable experimental data.

Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle

DEFF Research Database (Denmark)

Nyberg, Michael Permin; Al-Khazraji, Baraa K; Mortensen, Stefan P

2013-01-01

During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However...... studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1-10 µM) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition...... in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation....

Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

Science.gov (United States)

Niimi, Kenta; Ueda, Mizuha; Fukumoto, Moe; Kohara, Misaki; Sawano, Toshinori; Tsuchihashi, Ryo; Shibata, Satoshi; Inagaki, Shinobu; Furuyama, Tatsuo

2017-08-05

Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2012-12-15

Heating skin is believed to activate vanilloid type III and IV transient receptor potential ion channels (TRPV3, TRPV4, respectively), resulting in the release of ATP into the interstitial fluid. We examined the hypothesis that local skin heating would result in an accumulation of ATP in the interstitial fluid that would be related with a rise in skin blood flow (SkBF) and temperature sensation. Two microdialysis probes were inserted into the dermis on the dorsal aspect of the forearm in 15 young, healthy subjects. The probed skin was maintained at 31°C, 35°C, 39°C and 43°C for 8 min periods, during which SkBF was monitored as cutaneous vascular conductance (CVC). Dialysate was collected and analysed for ATP ([ATP](d)) using a luciferase-based assay, and ratings of perceived warmth were taken at each temperature. At a skin temperature of 31°C, [ATP](d) averaged 18.93 ± 4.06 nm and CVC averaged 12.57 ± 1.59% peak. Heating skin to 35°C resulted in an increase in CVC (17.63 ± 1.27% peak; P ATP](d). Heating skin to 39°C and 43°C resulted in a decreased [ATP](d) (5.88 ± 1.68 nm and 8.75 ± 3.44 nm, respectively; P ATP does not occur during local heating, and therefore does not have a role in temperature sensation or the dilator response in human skin. Nevertheless, the low threshold of dilatation (35°C) indicates a possible role for the TRPV3, TRPV4 channels or the sensitization of other ion channels in mediating the dilator response.

Characterizing the reflectivity of handheld display devices.

Science.gov (United States)

Liu, Peter; Badano, Aldo

2014-08-01

With increased use of handheld and tablet display devices for viewing medical images, methods for consistently measuring reflectivity of the devices are needed. In this note, the authors report on the characterization of diffuse reflections for handheld display devices including mobile phones and tablets using methods recommended by the American Association of Physicists in Medicine Task Group 18 (TG18). The authors modified the diffuse reflectance coefficient measurement method outlined in the TG18 report. The authors measured seven handheld display devices (two phones and five tablets) and three workstation displays. The device was attached to a black panel with Velcro. To study the effect of the back surface on the diffuse reflectance coefficient, the authors created Styrofoam masks with different size square openings and placed it in front of the device. Overall, for each display device, measurements of illuminance and reflected luminance on the display screen were taken. The authors measured with no mask, with masks of varying size, and with display-size masks, and calculated the corresponding diffuse reflectance coefficient. For all handhelds, the diffuse reflectance coefficient measured with no back panel were lower than measurements performed with a mask. The authors found an overall increase in reflectivity as the size of the mask decreases. For workstations displays, diffuse reflectance coefficients were higher when no back panel was used, and higher than with masks. In all cases, as luminance increased, illuminance increased, but not at the same rate. Since the size of handheld displays is smaller than that of workstation devices, the TG18 method suffers from a dependency on illumination condition. The authors show that the diffuse reflection coefficients can vary depending on the nature of the back surface of the illuminating box. The variability in the diffuse coefficient can be as large as 20% depending on the size of the mask. For all measurements

ATP-loading 201Tl myocardial SPECT for the detection of ischemic heart disease

International Nuclear Information System (INIS)

Fujinaga, Tsuyoshi; Yamazaki, Sayaka; Hara, Masatada; Umezawa, Chiaki; Okamura, Tetsuo; Murata, Hajime; Maruno, Hirotaka; Onoguchi, Masahisa.

1993-01-01

To evaluate the usefulness for the detection of ischemic heart disease, ATP myocrdial SPECT was performed in 35 patients (mean; 59±9.4 years) with angina pectoris or old myocardial infarction. Coronary angiography (CAG) was performed in all patients. The ultra-short half-life of ATP required a continuous infusion for its use. ATP was infused intravenously at a rate of 0.16 mg/kg/min for 5 min, with 201 Tl injection taking place at 3 min. Myocardial SPECT imaging was begun 5 min and 4 hr later after the end of ATP infusion. ATP caused a significant decrease in arterial blood pressure (p 201 Tl myocardial SPECT for the detection of coronary artery disease (CAD) was evaluated using CAG as a golden standard. The sensitivity and specificity for CAD detection were 82% and 90%, respectively. ATP myocardial SPECT is a promising new test for the detection of ischemic heart disease. (author)

Modeling complicated rheological behaviors in encapsulating shells of lipid-coated microbubbles accounting for nonlinear changes of both shell viscosity and elasticity.

Science.gov (United States)

Li, Qian; Matula, Thomas J; Tu, Juan; Guo, Xiasheng; Zhang, Dong

2013-02-21

It has been accepted that the dynamic responses of ultrasound contrast agent (UCA) microbubbles will be significantly affected by the encapsulating shell properties (e.g., shell elasticity and viscosity). In this work, a new model is proposed to describe the complicated rheological behaviors in an encapsulating shell of UCA microbubbles by applying the nonlinear 'Cross law' to the shell viscous term in the Marmottant model. The proposed new model was verified by fitting the dynamic responses of UCAs measured with either a high-speed optical imaging system or a light scattering system. The comparison results between the measured radius-time curves and the numerical simulations demonstrate that the 'compression-only' behavior of UCAs can be successfully simulated with the new model. Then, the shell elastic and viscous coefficients of SonoVue microbubbles were evaluated based on the new model simulations, and compared to the results obtained from some existing UCA models. The results confirm the capability of the current model for reducing the dependence of bubble shell parameters on the initial bubble radius, which indicates that the current model might be more comprehensive to describe the complex rheological nature (e.g., 'shear-thinning' and 'strain-softening') in encapsulating shells of UCA microbubbles by taking into account the nonlinear changes of both shell elasticity and shell viscosity.

Modeling complicated rheological behaviors in encapsulating shells of lipid-coated microbubbles accounting for nonlinear changes of both shell viscosity and elasticity

International Nuclear Information System (INIS)

Li Qian; Tu Juan; Guo Xiasheng; Zhang Dong; Matula, Thomas J

2013-01-01

It has been accepted that the dynamic responses of ultrasound contrast agent (UCA) microbubbles will be significantly affected by the encapsulating shell properties (e.g., shell elasticity and viscosity). In this work, a new model is proposed to describe the complicated rheological behaviors in an encapsulating shell of UCA microbubbles by applying the nonlinear ‘Cross law’ to the shell viscous term in the Marmottant model. The proposed new model was verified by fitting the dynamic responses of UCAs measured with either a high-speed optical imaging system or a light scattering system. The comparison results between the measured radius–time curves and the numerical simulations demonstrate that the ‘compression-only’ behavior of UCAs can be successfully simulated with the new model. Then, the shell elastic and viscous coefficients of SonoVue microbubbles were evaluated based on the new model simulations, and compared to the results obtained from some existing UCA models. The results confirm the capability of the current model for reducing the dependence of bubble shell parameters on the initial bubble radius, which indicates that the current model might be more comprehensive to describe the complex rheological nature (e.g., ‘shear-thinning’ and ‘strain-softening’) in encapsulating shells of UCA microbubbles by taking into account the nonlinear changes of both shell elasticity and shell viscosity. (paper)

Molecular mechanism of bacterial Hsp90 pH-dependent ATPase activity.

Science.gov (United States)

Jin, Yi; Hoxie, Reyal S; Street, Timothy O

2017-06-01

Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated open-to-closed-to-open conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pH-dependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformation-specific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anti-correlation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255-ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255-ATP salt-bridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pH-dependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal site-specific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity. © 2017 The Protein Society.

Intracellular ATP influences synaptic plasticity in area CA1 of rat hippocampus via metabolism to adenosine and activity-dependent activation of adenosine A1 receptors.

Science.gov (United States)

zur Nedden, Stephanie; Hawley, Simon; Pentland, Naomi; Hardie, D Grahame; Doney, Alexander S; Frenguelli, Bruno G

2011-04-20

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to ≥ 0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with d-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A(1) receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

DEFF Research Database (Denmark)

Liu, Jianming; Kandasamy, Vijayalakshmi; Würtz, Anders

2016-01-01

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase into a Lact...

Nonlinear response of ultrasound contrast agent microbubbles: From fundamentals to applications

International Nuclear Information System (INIS)

Teng Xu-Dong; Guo Xia-Sheng; Tu Juan; Zhang Dong

2016-01-01

Modelling and biomedical applications of ultrasound contrast agent (UCA) microbubbles have attracted a great deal of attention. In this review, we summarize a series of researches done in our group, including (i) the development of an all-in-one solution of characterizing coated bubble parameters based on the light scattering technique and flow cytometry; (ii) a novel bubble dynamic model that takes into consideration both nonlinear shell elasticity and viscosity to eliminate the dependences of bubble shell parameters on bubble size; (iii) the evaluation of UCA inertial cavitation threshold and its relationship with shell parameters; and (iv) the investigations of transfection efficiency and the reduction of cytotoxicity in gene delivery facilitated by UCAs excited by ultrasound exposures. (special topic)

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

Directory of Open Access Journals (Sweden)

Patrick E MacDonald

2007-06-01

Full Text Available Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+ responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+ signalling was blocked, but was reversed by low concentrations (1-20 muM of the ATP-sensitive K(+ (KATP channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM. Higher diazoxide concentrations (>/=30 muM decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM, glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+ (TTX and N-type Ca(2+ channels (omega-conotoxin, but not L-type Ca(2+ channels (nifedipine, prevented glucagon secretion. Both the N-type Ca(2+ channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch

Directory of Open Access Journals (Sweden)

Steven Zhao

2016-10-01

Full Text Available Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY, cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch.

Science.gov (United States)

Zhao, Steven; Torres, AnnMarie; Henry, Ryan A; Trefely, Sophie; Wallace, Martina; Lee, Joyce V; Carrer, Alessandro; Sengupta, Arjun; Campbell, Sydney L; Kuo, Yin-Ming; Frey, Alexander J; Meurs, Noah; Viola, John M; Blair, Ian A; Weljie, Aalim M; Metallo, Christian M; Snyder, Nathaniel W; Andrews, Andrew J; Wellen, Kathryn E

2016-10-18

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A new species of fiddler crab from the Ogasawara (Bonin) Islands, Japan, separated from the widely-distributed sister species Uca (Paraleptuca) crassipes (White, 1847) (Crustacea: Decapoda: Brachyura: Ocypodidae).

Science.gov (United States)

Shih, Hsi-Te; Komai, Tomoyuki; Liu, Min-Yun

2013-12-10

A new species of fiddler crab (Brachyura: Ocypodidae), Uca boninensis sp. nov., is described from the Ogasawara (Bonin) Islands, Japan. The new species has previously been identified with the widely distributed U. crassipes (White, 1847), from which it differs by having a slightly differently shaped carapace, and relatively stouter male first gonopods (G1). The recognition of the new species is also supported by differences in the mitochondrial cytochrome oxidase I (COI) and control region (CR) genes. U. boninensis sp. nov., appears to be endemic to the Ogasawara Islands, and as the only known population is small, urgent conservation measures are needed for its protection. Our study brings the total number of the Japanese fiddler crab species to 12.

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

NARCIS (Netherlands)

Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C. D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

2014-01-01

Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we

Evaluation of the in vitro Chemosensitivity and Correlation with Clinical Outcomes in Lung Cancer using the ATP-TCA.

Science.gov (United States)

Chen, Zhiyao; Zhang, Shichao; Ma, Sheng; Li, Chang; Xu, Chun; Shen, Yinfang; Zhao, Jun; Miao, Liyan

2018-01-01

Multiple drug resistance (MDR) to chemotherapeutic agents often leads to a failure to respond to chemotherapy. We utilized an in vitro chemosensitivity test to identify sensitive and effective chemotherapeutic drugs and further elucidated the correlation between the in vivo chemosensitivity and clinical outcomes. Here, we evaluated the in vitro chemosensitivity and MDR of 120 lung cancer patients to eight singledrug chemotherapies and of 291 lung cancer patients to seven chemotherapy regimens using an ATP-based tumor chemosensitivity assay (ATP-TCA). Additionally, the chemosensitivity profiles of lung adenocarcinoma patients (284 cases) and lung squamous cell carcinoma patients (90 cases) to these single-drug and chemotherapy regimens were compared. Furthermore, the correlations between the chemosensitivity and clinical outcomes were investigated in 16 stage III squamous cell carcinoma patients. PTX (51.7%), TXT (43.3%), GEM (12.5%), PTX+DDP (62.5%), TXT+L-OHP (54.3%) and VP-16+DDP (16.2%) had the highest in vitro chemosensitivity rates. Approximately 31.7% of patients developed resistance to all eight single-drug chemotherapies, and 25.8% of patients displayed resistance to all seven chemotherapy regimens. In addition, lung squamous cell carcinoma was significantly more sensitive to GEM and MTA+DDP than lung adenocarcinoma (P<0.05). Further analysis showed that patients with higher drug sensitivity tended to have longer disease-free survival (18 months vs. 8.5 months) than patients displaying drug resistance (P<0.05). These results suggest that the implementation of in vitro drug susceptibility testing before chemotherapy can effectively prevent the occurrence of primary drug resistance and inappropriate drug treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A vanadium-dependent bromoperoxidase in the marine red alga Kappaphycus alvarezii (Doty) Doty displays clear substrate specificity.

Science.gov (United States)

Kamenarska, Zornitsa; Taniguchi, Tomokazu; Ohsawa, Noboru; Hiraoka, Masanori; Itoh, Nobuya

2007-05-01

Bromoperoxidase activity was initially detected in marine macroalgae belonging to the Solieriaceae family (Gigartinales, Rhodophyta), including Solieria robusta (Greville) Kylin, Eucheuma serra J. Agardh and Kappaphycus alvarezii (Doty) Doty, which are important industrial sources of the polysaccharide carrageenan. Notably, the purification of bromoperoxidase was difficult because due to the coexistence of viscoid polysaccharides. The activity of the partially purified enzyme was dependent on the vanadate ion, and displayed a distinct substrate spectrum from that of previously reported vanadium-dependent bromoperoxidases of marine macroalgae. The enzyme was specific for Br- and I- ions and inactive toward F- and Cl-. The K(m) values for Br- and H2O2 were 2.5x10(-3) M and 8.5x10(-5) M, respectively. The halogenated product, dibromoacetaldehyde, that accumulated in K. alvarezii was additionally determined.

Low-level light therapy potentiates NPe6-mediated photodynamic therapy in a human osteosarcoma cell line via increased ATP.

Science.gov (United States)

Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R

2015-03-01

Low-level light therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-l-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5J/cm(2) of 810nm near infrared radiation (NIR) followed by addition of 10μM NPe6 and after 2h incubation by 1.5J/cm(2) of 652nm red light for PDT. PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of luciferase probes to measure ATP in living cells and animals.

Science.gov (United States)

Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

2017-08-01

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

Label free luminescence strategy for sensitive detection of ATP using aptamer-Ru(II) complexes

Energy Technology Data Exchange (ETDEWEB)

Babu, Eththilu [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Muthu Mareeswaran, Paulpandian [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Department of Industrial Chemistry, Alagappa Univesity, Karaikudi 630003, Tamil Nadu (India); Ramdass, Arumugam [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Research Department of Chemistry, Aditanar College of Arts and Science, Tiruchendur 628216, Tamil Nadu (India); Ramesh, Pandian [UCIBIO-REQUIMTE, Departmento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica (Portugal); Rajagopal, Seenivasan, E-mail: rajagopalseenivasan@yahoo.com [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India)

2016-07-15

A simple and sensitive aptamer-based luminescence strategy for ATP detection is developed using Ru(II) complexes as probe molecule. It is based on the fact that Ru(II)-dppz complexes show the light switching behavior with DNA aptamers and found to show significant luminescence spectral change on the addition of ATP molecules. The binding efficiencies of aptamer with ATP, ADP and AMP are calculated and compared. The structural change of aptamer is also studied using circular dichroism (CD) spectral techniques. Moreover, the binding nature of aptamer with ATP, ADP and AMP is demonstrated by computational techniques. The proposed strategy was successfully applied to the detection of ATP.

Label free luminescence strategy for sensitive detection of ATP using aptamer-Ru(II) complexes

International Nuclear Information System (INIS)

Babu, Eththilu; Muthu Mareeswaran, Paulpandian; Ramdass, Arumugam; Ramesh, Pandian; Rajagopal, Seenivasan

2016-01-01

A simple and sensitive aptamer-based luminescence strategy for ATP detection is developed using Ru(II) complexes as probe molecule. It is based on the fact that Ru(II)-dppz complexes show the light switching behavior with DNA aptamers and found to show significant luminescence spectral change on the addition of ATP molecules. The binding efficiencies of aptamer with ATP, ADP and AMP are calculated and compared. The structural change of aptamer is also studied using circular dichroism (CD) spectral techniques. Moreover, the binding nature of aptamer with ATP, ADP and AMP is demonstrated by computational techniques. The proposed strategy was successfully applied to the detection of ATP.

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

Science.gov (United States)

Heinzen, Erin L.; Swoboda, Kathryn J.; Hitomi, Yuki; Gurrieri, Fiorella; Nicole, Sophie; de Vries, Boukje; Tiziano, F. Danilo; Fontaine, Bertrand; Walley, Nicole M.; Heavin, Sinéad; Panagiotakaki, Eleni; Fiori, Stefania; Abiusi, Emanuela; Di Pietro, Lorena; Sweney, Matthew T.; Newcomb, Tara M.; Viollet, Louis; Huff, Chad; Jorde, Lynn B.; Reyna, Sandra P.; Murphy, Kelley J.; Shianna, Kevin V.; Gumbs, Curtis E.; Little, Latasha; Silver, Kenneth; Ptác̆ek, Louis J.; Haan, Joost; Ferrari, Michel D.; Bye, Ann M.; Herkes, Geoffrey K.; Whitelaw, Charlotte M.; Webb, David; Lynch, Bryan J.; Uldall, Peter; King, Mary D.; Scheffer, Ingrid E.; Neri, Giovanni; Arzimanoglou, Alexis; van den Maagdenberg, Arn M.J.M.; Sisodiya, Sanjay M.; Mikati, Mohamad A.; Goldstein, David B.; Nicole, Sophie; Gurrieri, Fiorella; Neri, Giovanni; de Vries, Boukje; Koelewijn, Stephany; Kamphorst, Jessica; Geilenkirchen, Marije; Pelzer, Nadine; Laan, Laura; Haan, Joost; Ferrari, Michel; van den Maagdenberg, Arn; Zucca, Claudio; Bassi, Maria Teresa; Franchini, Filippo; Vavassori, Rosaria; Giannotta, Melania; Gobbi, Giuseppe; Granata, Tiziana; Nardocci, Nardo; De Grandis, Elisa; Veneselli, Edvige; Stagnaro, Michela; Gurrieri, Fiorella; Neri, Giovanni; Vigevano, Federico; Panagiotakaki, Eleni; Oechsler, Claudia; Arzimanoglou, Alexis; Nicole, Sophie; Giannotta, Melania; Gobbi, Giuseppe; Ninan, Miriam; Neville, Brian; Ebinger, Friedrich; Fons, Carmen; Campistol, Jaume; Kemlink, David; Nevsimalova, Sona; Laan, Laura; Peeters-Scholte, Cacha; van den Maagdenberg, Arn; Casaer, Paul; Casari, Giorgio; Sange, Guenter; Spiel, Georg; Boneschi, Filippo Martinelli; Zucca, Claudio; Bassi, Maria Teresa; Schyns, Tsveta; Crawley, Francis; Poncelin, Dominique; Vavassori, Rosaria

2012-01-01

Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurologic manifestations. AHC is usually a sporadic disorder with unknown etiology. Using exome sequencing of seven patients with AHC, and their unaffected parents, we identified de novo nonsynonymous mutations in ATP1A3 in all seven AHC patients. Subsequent sequence analysis of ATP1A3 in 98 additional patients revealed that 78% of AHC cases have a likely causal ATP1A3 mutation, including one inherited mutation in a familial case of AHC. Remarkably, six ATP1A3 mutations explain the majority of patients, including one observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset-dystonia-parkinsonism, AHC-causing mutations revealed consistent reductions in ATPase activity without effects on protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC, and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in this gene. PMID:22842232

Tumour exosomes display different differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness

DEFF Research Database (Denmark)

Whitehead, Bradley Joseph; Wu, Linping; Hvam, Michael Lykke

2015-01-01

(QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement......). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner. Conclusions : Malignant (metastatic and non-metastatic) cell line exosomes display a markedly reduced stiffness and adhesion but an increased complement activation compared to non...

Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

Science.gov (United States)

Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

2010-03-05

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

Science.gov (United States)

He, Jiuya; Ford, Holly C; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2017-03-28

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1 , ATP5G2 , and ATP5G3 , encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1 , ATP5G2 , and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

Science.gov (United States)

Vandenbeuch, Aurelie; Anderson, Catherine B; Kinnamon, Sue C

2015-09-01

Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.

Science.gov (United States)

Vázquez-Cuevas, Francisco G; Martínez-Ramírez, Angélica S; Robles-Martínez, Leticia; Garay, Edith; García-Carrancá, Alejandro; Pérez-Montiel, Delia; Castañeda-García, Carolina; Arellano, Rogelio O

2014-11-01

P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability. © 2014 Wiley Periodicals, Inc.

Mesurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Ytting, Cecilie Karkov; Fuglsang, Anja Thoe; Hiltunen, J. Kalervo

2012-01-01

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating...... of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal...... activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose...

The Contribution of Red Blood Cell Dynamics to Intrinsic Viscosity and Functional ATP Release

Science.gov (United States)

Forsyth, Alison; Abkarian, Manouk; Wan, Jiandi; Stone, Howard

2010-11-01

In shear flow, red blood cells (RBCs) exhibit a variety of behaviors such as rouleaux formation, tumbling, swinging, and tank-treading. The physiological consequences of these dynamic behaviors are not understood. In vivo, ATP is known to signal vasodilation; however, to our knowledge, no one has deciphered the relevance of RBC microrheology to the functional release of ATP. Previously, we correlated RBC deformation and ATP release in microfluidic constrictions (Wan et al., 2008). In this work, a cone-plate rheometer is used to shear a low hematocrit solution of RBCs at varying viscosity ratios (λ) between the inner cytoplasmic hemoglobin and the outer medium, to determine the intrinsic viscosity of the suspension. Further, using a luciferin-luciferase enzymatic reaction, we report the relative ATP release at varying shear rates. Results indicate that for λ = 1.6, 3.8 and 11.1, ATP release is constant up to 500 s-1, which suggests that the tumbling-tanktreading transition does not alter ATP release in pure shear. For lower viscosity ratios, λ = 1.6 and 3.8, at 500 s-1 a change in slope occurs in the intrinsic viscosity data and is marked by an increase in ATP release. Based on microfluidic observations, this simultaneous change in viscosity and ATP release occurs within the tank-treading regime.

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion.

Science.gov (United States)

Cascales, Eric; Christie, Peter J

2004-12-07

Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa. Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate [T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] of the Agrobacterium tumefaciens VirB/D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel. Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility. VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation. Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9. We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 "bridge" between inner and outer membrane subassemblies of the VirB/D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope.

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

Directory of Open Access Journals (Sweden)

Olga B. Vadzyuk

2018-03-01

Full Text Available The objective of this study was to detect ATP-sensitive K+ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K+-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of KATP-channel (ATP-sensitive K+-channel – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate. Our data demonstrates that K+ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of KATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of KATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane. Keywords: ATP-sensitive potassium channel, Diazoxide, 5-hydroxydecanoate, Myometrium, Mitochondria, Mitochondrial swelling, Mitochondrial membrane potential, ROS

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

International Nuclear Information System (INIS)

Kasho, V.N.; Boyer, P.D.

1989-01-01

Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F 1 , F 0 -ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F 1 ,F 0 -ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P i as medium ATP concentration was lowered was determined by 18 O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P i formed as ATP concentration is decreased to the micromolar range. The F 1 ,F 0 -ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F 1 -type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F 1 ,F 0 -ATPases

Rapid granulation tissue regeneration by intracellular ATP delivery--a comparison with Regranex.

Directory of Open Access Journals (Sweden)

Jeffrey D Howard

Full Text Available This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.

Role of ATP in the regulation of cholesterol biogenesis

International Nuclear Information System (INIS)

Subba Rao, G.; Ramasarma, T.

1974-01-01

Intraperitoneal administration of glucose (4oomg/rat) stimulated the biogenesis of sterols in starved rats while citrate or pyruvate (20mg/rat) did not have any effect. ATP (10mg/ rat) administered intraperitoneally stimulated the incorporation of acetate-1- 14 C into sterols but not of mevalonate-2- 14 C into sterols in starved rats. The results indicate that ATP may play a role in regulating cholesterol biogenesis and it is not acting merely as an energy source. (author)

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

International Nuclear Information System (INIS)

Ahluwalia, Unnati; Deep, Shashank

2012-01-01

Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

Display Rules for Anger and Aggression in School-Age Children.

Science.gov (United States)

Underwood, Marion K.; And Others

1992-01-01

Two studies examined the development of display rules for anger and the relationship between the use of display rules for anger and aggressiveness as rated by school peers. Findings indicate that the phenomenon of display rules for anger is complex and depends on the way display rules are defined and the age and gender of the subjects. (GLR)

Quantum-mechanical analysis of amino acid residues function in the proton transport during F0F1-ATP synthase catalytic cycle

Science.gov (United States)

Ivontsin, L. A.; Mashkovtseva, E. V.; Nartsissov, Ya R.

2017-11-01

Implications of quantum-mechanical approach to the description of proton transport in biological systems are a tempting subject for an overlapping of fundamental physics and biology. The model of proton transport through the integrated membrane enzyme FoF1-ATP synthase responsible for ATP synthesis was developed. The estimation of the mathematical expectation of the proton transfer time through the half-channel was performed. Observed set of proton pathways through the inlet half-channel showed the nanosecond timescale highly dependable of some amino acid residues. There were proposed two types of crucial amino acids: critically localized (His245) and being a part of energy conserving system (Asp119).

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

To date, it is unknown whether mitochondrial dysfunction in skeletal muscle from subjects with type 2 diabetes is based on primarily reduced mitochondrial mass and/or a primarily decreased mitochondrial ATP synthesis. Mitochondrial mass were determined in myotubes established from eight lean, eight...... 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...... 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...

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Science.gov (United States)

Thakur, Manoj; Kumar, Mohan B J; Muniyappa, K

2016-10-18

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

ADP-dependent Phosphofructokinases in Mesophilic and Thermophilic Methanogenic Archaea

NARCIS (Netherlands)

Verhees, C.H.; Tuininga, J.E.; Kengen, S.W.M.; Stams, A.J.M.; Oost, van der J.; Vos, de W.M.

2001-01-01

Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PPi-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus

Neonatal diabetes and congenital hyperinsulinism caused by mutations in ABCC8/SUR1 are associated with altered and opposite affinities for ATP and ADP

Directory of Open Access Journals (Sweden)

Joseph eBryan

2015-04-01

Full Text Available ATP-sensitive K+ (KATP channels composed of potassium inward-rectifier type 6.2 and sulfonylurea receptor type 1 subunits (Kir6.2/SUR14 are expressed in various cells in the brain and endocrine pancreas where they couple metabolic status to membrane potential. In β-cells, increases in cytosolic [ATP/ADP]c inhibit KATP channel activity, leading to membrane depolarization and exocytosis of insulin granules. Mutations in ABCC8 (SUR1 or KCNJ11 (Kir6.2 can result in gain or loss of channel activity and cause neonatal diabetes (ND or congenital hyperinsulinism (CHI, respectively. SUR1 is reported to be a Mg2+-dependent ATPase. A prevailing model posits that ATP hydrolysis at SUR1 is required to stimulate openings of the pore. However, recent work shows nucleotide binding, without hydrolysis, is sufficient to switch SUR1 to stimulatory conformations. The actions of nucleotides, ATP and ADP, on ND (SUR1E1506D and CHI (SUR1E1506K mutants, without Kir6.2, were compared to assess both models. Both substitutions significantly impair hydrolysis in SUR1 homologues. SUR1E1506D has greater affinity for MgATP than wildtype; SUR1E1506K has reduced affinity. Without Mg2+, SUR1E1506K has a greater affinity for ATP4- consistent with electrostatic attraction between ATP4-, unshielded by Mg2+, and the basic lysine. Further analysis of ND and CHI ABCC8 mutants in the second transmembrane and nucleotide binding domains (TMD2 & NBD2, found a relation between their affinities for ATP (± Mg2+ and their clinical phenotype. Increased affinity for ATP is associated with ND; decreased affinity with CHI. In contrast, MgADP showed a weaker relationship. Diazoxide, known to reduce insulin release in some CHI cases, potentiates switching of CHI mutants from non-stimulatory to stimulatory states consistent with diazoxide stabilizing a nucleotide-bound conformation. The results emphasize the greater importance of nucleotide binding vs hydrolysis in the regulation of KATP channels

The Effects of Advanced 'Glass Cockpit' Displayed Flight Instrumentation on In-flight Pilot Decision Making

Science.gov (United States)

Steigerwald, John

The Cognitive Continuum Theory (CCT) was first proposed 25 years ago to explain the relationship between intuition and analytical decision making processes. In order for aircraft pilots to make these analytical and intuitive decisions, they obtain information from various instruments within the cockpit of the aircraft. Advanced instrumentation is used to provide a broad array of information about the aircraft condition and flight situation to aid the flight crew in making effective decisions. The problem addressed is that advanced instrumentation has not improved the pilot decision making in modern aircraft. Because making a decision is dependent upon the information available, this experimental quantitative study sought to determine how well pilots organize and interpret information obtained from various cockpit instrumentation displays when under time pressure. The population for this study was the students, flight instructors, and aviation faculty at the Middle Georgia State College School of Aviation campus in Eastman, Georgia. The sample was comprised of two groups of 90 individuals (45 in each group) in various stages of pilot licensure from student pilot to airline transport pilot (ATP). The ages ranged from 18 to 55 years old. There was a statistically significant relationship at the p safety of flight.

Gauging the likelihood of stable cavitation from ultrasound contrast agents.

Science.gov (United States)

Bader, Kenneth B; Holland, Christy K

2013-01-07

The mechanical index (MI) was formulated to gauge the likelihood of adverse bioeffects from inertial cavitation. However, the MI formulation did not consider bubble activity from stable cavitation. This type of bubble activity can be readily nucleated from ultrasound contrast agents (UCAs) and has the potential to promote beneficial bioeffects. Here, the presence of stable cavitation is determined numerically by tracking the onset of subharmonic oscillations within a population of bubbles for frequencies up to 7 MHz and peak rarefactional pressures up to 3 MPa. In addition, the acoustic pressure rupture threshold of an UCA population was determined using the Marmottant model. The threshold for subharmonic emissions of optimally sized bubbles was found to be lower than the inertial cavitation threshold for all frequencies studied. The rupture thresholds of optimally sized UCAs were found to be lower than the threshold for subharmonic emissions for either single cycle or steady state acoustic excitations. Because the thresholds of both subharmonic emissions and UCA rupture are linearly dependent on frequency, an index of the form I(CAV) = P(r)/f (where P(r) is the peak rarefactional pressure in MPa and f is the frequency in MHz) was derived to gauge the likelihood of subharmonic emissions due to stable cavitation activity nucleated from UCAs.

Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells.

Science.gov (United States)

Deng, Jingjing; Wang, Kai; Wang, Ming; Yu, Ping; Mao, Lanqun

2017-04-26

Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn 2+ and imidazole-2-carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

High-altitude adaptation of Tibetan chicken from MT-COI and ATP-6 perspective.

Science.gov (United States)

Zhao, Xiaoling; Wu, Nan; Zhu, Qing; Gaur, Uma; Gu, Ting; Li, Diyan

2016-09-01

The problem of hypoxia adaptation in high altitudes is an unsolved brainteaser in the field of life sciences. As one of the best chicken breeds with adaptability to highland environment, the Tibetan chicken, is genetically different from lowland chicken breeds. In order to gain a better understanding of the mechanism of hypoxic adaptability in high altitude, in the present study, we focused on the MT-COI together with ATP-6 gene to explore the regulatory mechanisms for hypoxia adaptability in Tibet chicken. Here, we sequenced MT-COI of 29 Tibetan chickens and 30 Chinese domestic chickens and ATP-6 gene of 28 Tibetan chickens and 29 Chinese domestic chickens. In MT-COI gene, 9 single nucleotide polymorphisms (SNPs) were detected though none of these was a missense mutation, confirming the fact that MT-COI gene is a largely conservative sequence. In ATP-6 gene, 6 single nucleotide polymorphisms (SNPs) were detected and we found a missense mutation (m.9441G > A) in the ATP-6 gene of Tibetan chicken resulting in an amino acid substitution. Due to the critical role of ATP-6 gene in the proton translocation and energy metabolism, we speculated the possibility of this mutation playing an important role in easier energy conversion and metabolism in Tibetan chickens than Chinese domestic chickens so as to better adapt to the harsh environment of the high-altitude areas. The Median-joining profile also suggested that haplotype Ha2 has the ancestral position to the other haplotypes and has significant relationship with high-altitude adaptation in ATP-6 gene. Therefore, we considered that the polymorphism (m.9441G > A) in the ATP-6 gene may affect the specific functions of ATP-6 enzyme relating to high-altitude adaptation of Tibetan chicken and MT-COI gene is a largely conservative sequence.

Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.

Science.gov (United States)

Sugimoto, H; Ikeda, K; Kawakami, K

2017-10-23

Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3 +/- ) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3 +/- and compared the rank and hierarchy structure between Atp1a3 +/- and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3 +/- than wild-type mice, and Atp1a3 +/- approached Atp1a3 +/- mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3 +/- group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3 +/- and wild type. (4) Atp1a3 +/- showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3 +/- showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

A two-hour antibiotic susceptibility test by ATP-bioluminescence.

Science.gov (United States)

March Rosselló, Gabriel Alberto; García-Loygorri Jordán de Urries, María Cristina; Gutiérrez Rodríguez, María Purificación; Simarro Grande, María; Orduña Domingo, Antonio; Bratos Pérez, Miguel Ángel

2016-01-01

The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Preferred Color Gamut Boundaries for Wide-Gamut and Multi-Primary Displays

NARCIS (Netherlands)

Murdoch, M.J.; Sekulovski, S.D.; Heynderickx, I.E.J.

2012-01-01

Preferred chroma enhancement and its dependence on hue were studiedin a two-part experiment using a wide-gamut multi-primary display.This builds on earlier experiments which showed a clear hue dependence but were limited by the gamut of the display they employed. In the first part of this

Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.

Science.gov (United States)

Dey, Sanjay; Biswas, Maitree; Sen, Udayaditya; Dasgupta, Jhimli

2015-04-03

Bacterial enhancer-binding proteins (bEBPs) oligomerize through AAA(+) domains and use ATP hydrolysis-driven energy to isomerize the RNA polymerase-σ(54) complex during transcriptional initiation. Here, we describe the first structure of the central AAA(+) domain of the flagellar regulatory protein FlrC (FlrC(C)), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrC(C) forms heptamer both in nucleotide (Nt)-free and -bound states without ATP-dependent subunit remodeling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated "all or none" ATP binding occurs in the heptameric FlrC(C), because constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of the trans-protomer required for Nt binding. A unique "closed to open" movement of Walker A, assisted by trans-acting "Glu switch" Glu-286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrC(C) and mutants revealed that although Arg-349 of sensor II, positioned by trans-acting Glu-286 and Tyr-290, acts as a key residue to bind and hydrolyze ATP, Arg-319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrC(C) is restored in solution even with the transition state mimicking ADP·AlF3. Structural results and pulldown assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ(54)-FlrC(C) interaction independent of Nt binding. Collectively, our results underscore a novel mechanism of ATP binding and σ(54) interaction that strives to understand the transcriptional mechanism of the bEBPs, which probably interact directly with the RNA polymerase-σ(54) complex without DNA looping. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cloning and characterization of ATP synthase CF1 α gene from ...

African Journals Online (AJOL)

ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels.

NARCIS (Netherlands)

Graaf, A.O. de; Meijerink, J.P.P.; Heuvel, L.P.W.J. van den; Abreu, R.A. de; Witte, T.J.M. de; Jansen, J.H.; Smeitink, J.A.M.

2002-01-01

Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production

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

Energy Technology Data Exchange (ETDEWEB)

Celikel, Reha; Veldore, Vidya Harini [University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Mathews, Irimpan [Stanford Synchrotron Radiation Lightsource, 2575 Sand Hill Road, Menlo Park, CA 94025 (United States); Devine, Kevin M., E-mail: kdevine@tcd.ie [Trinity College Dublin, Dublin 2 (Ireland); Varughese, Kottayil I., E-mail: kdevine@tcd.ie [University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

2012-07-01

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 Mg{sup 2+} 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.

Redox regulation of ATP sulfurylase in microalgae

Czech Academy of Sciences Publication Activity Database

Prioretti, L.; Lebrun, R.; Gontero, B.; Giordano, Mario

2016-01-01

Roč. 478, č. 4 (2016), s. 1555-1562 ISSN 0006-291X Institutional support: RVO:61388971 Keywords : ATP sulfurylase * cysteine * Sulfur metabolism Subject RIV: EE - Microbiology, Virology Impact factor: 2.466, year: 2016

Intestinal bile salt absorption in Atp8b1 deficient mice

NARCIS (Netherlands)

Groen, Annemiek; Kunne, Cindy; Paulusma, Coen C.; Kramer, Werner; Agellon, Luis B.; Bull, Laura N.; Elferink, Ronald P. J. Oude

2007-01-01

BACKGROUND/AIMS: Mutations in the ATP8B1 gene can cause Progressive Familial Intrahepatic Cholestasis type 1. We have previously reported that Atp8b1(G308V/G308V) mice, a model for PFIC1, have slightly, but significantly, higher baseline serum bile salt (BS) concentrations compared to wt mice. Upon

Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

Directory of Open Access Journals (Sweden)

David A. Dunn

2012-01-01

Full Text Available Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M or wildtype (A6W mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P0.05. This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

p.H1069Q mutation in ATP7B and biochemical parameters of copper metabolism and clinical manifestation of Wilson's disease.

Science.gov (United States)

Gromadzka, Graznya; Schmidt, Harmut H J; Genschel, Janine; Bochow, Bettina; Rodo, M; Tarnacka, Beatek; Litwin, Thomas; Chabik, Grzegorz; Członkowska, Anna

2006-02-01

We compared the effect of the p.H1069Q mutation and other non-p.H1069Q mutations in ATP7B on the phenotypic expression of Wilson's disease (WD), and assessed whether the clinical phenotype of WD in compound heterozygotes depends on the type of mutation coexisting with the p.H1069Q. One hundred forty-two patients with clinically, biochemically, and genetically diagnosed WD were studied. The mutational analysis of ATP7B was performed by direct sequencing. A total number of 26 mutations in ATP7B were identified. The p.His1069Gln was the most common mutation (allelic frequency: 72%). Seventy-three patients were homozygous for this mutation. Of compound heterozygotes, 37 had frameshift/nonsense mutation, and 20 had other missense mutation on one of their ATP7B alleles. Twelve patients had two non-p.H1069Q mutations. Patients homozygous for the p.H1069Q mutation had the less severe disturbances of copper metabolism and the latest presentation of first WD symptoms. The most severely disturbed copper metabolism and the earliest age at initial disease manifestation was noticed in non-p.H1069Q patients. In compound heterozygotes, the type of mutation coexisting with the p.H1069Q to a small extent influenced WD phenotype. The phenotype of WD varied considerably among patients with the same genotype. The p.H1069Q mutation is associated with late WD manifestation and with a mild disruption of copper metabolism. In compound heterozygotes, the phenotype of WD to a small extent depends on the type of mutation coexisting with the p.H1069Q. Besides genotype, additional modifying factors seem to determine WD manifestations. Copyright (c) 2005 Movement Disorder Society.

Structural models of zebrafish (Danio rerio NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

Directory of Open Access Journals (Sweden)

Jitendra Maharana

Full Text Available Nucleotide-binding oligomerization domain-containing protein 1 (NOD1 and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds and Aspartic acid (Walker B formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2 interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Long-Range Effects of Na(+) Binding in Na,K-ATPase Reported by ATP.

Science.gov (United States)

Middleton, David A; Fedosova, Natalya U; Esmann, Mikael

2015-12-01

This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

International Nuclear Information System (INIS)

Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

2013-01-01

Highlights: ► ATP-treated sciatic explants shows the decreased expression of p75NGFR. ► Extracellular ATP inhibits the expression of phospho-ERK1/2. ► Lysosomal exocytosis is involved in Schwann cell dedifferentiation. ► Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes

Directory of Open Access Journals (Sweden)

Francesco Drago

2017-12-01

Full Text Available Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs, which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012. However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.

Functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid

International Nuclear Information System (INIS)

Rothenberg, M.; Hanson, M.R.

1988-01-01

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences using electrophoresis and autoradiography, indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts

Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

Science.gov (United States)

Jastreboff, M; Kedzierska, B; Rode, W

1982-01-15

Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.

Comparação da estrutura populacional das espécies de Uca (Crustacea: Decapoda: Ocypodidae no Manguezal de Itacuruçá, Rio de Janeiro, Brasil Comparison of the population structure of the species of Uca (Crustacea: Decapoda: Ocypodidae in the mangrove of Itacuruçá, Rio Janeiro, Brazil

Directory of Open Access Journals (Sweden)

Luciane M. Bedê

2008-12-01

Full Text Available Este trabalho foi realizado no Manguezal de Itacuruçá, na Baía de Sepetiba com o objetivo de analisar a estrutura populacional das espécies de Uca Leach, 1814. Foram realizadas coletas de junho/2005 a maio/2006, durante as marés baixas. Os caranguejos foram capturados manualmente por duas pessoas e durante 15 minutos. Um total de 2580 animais foi coletado, sendo 1465 machos e 1115 fêmeas. Com relação ao tamanho dos indivíduos, observou-se que os animais do Manguezal de Itacuruçá, de maneira geral, apresentam tamanhos menores que os encontrados em outros manguezais do Brasil. Contudo, os machos atingiram tamanhos maiores do que as fêmeas. A distribuição de freqüência em todas as classes de tamanho foi unimodal para a maioria das espécies, com exceção de U. thayeri Rathbun, 1900 e U. vocator (Herbst, 1804, as quais não apresentaram um padrão definido. Os machos foram mais abundantes em todas as classes de maiores tamanhos. A razão sexual diferiu significativamente da proporção 1:1, estando deslocada para uma maior freqüência de machos, com exceção de U. thayeri e U. victoriana von Hagen, 1987, as quais tiveram predominância de fêmeas.This study was conducted in the Itacuruçá mangrove, in the Sepetiba Bay, Rio de Janeiro, Brazil, with the objective of investigating the population structure of the species of Uca Leach, 1814. Sampling was carried out monthly from June, 2005 to May, 2006, during low tides. Crabs were captured manually by two people for a period of 15 min each. A total of 2580 crabs were obtained, of which 1465 were males and 1115 were females. The size of the fiddler crabs in the mangrove of Itacuruçá were the smallest reported so far in Brazilian mangroves. However, males attained a larger size than females. The size frequency distribution was unimodal for most of species, with the exception of U. thayeri Rathbun, 1900 and U. vocator (Herbst, 1804, which did not show a clearly-defined pattern. Males

Degradation of ATP and glycogen in cod ( Gadus morhua ) muscle during freezing

DEFF Research Database (Denmark)

Cappeln, Gertrud; Jessen, Flemming

2001-01-01

Changes in ATP, IMP, lactate and glycogen contents in the muscle of cod were followed during freezing at temperatures of -20C and -45C. ATP degradation was accompanied by a corresponding increase in IMP content. Simultaneous measurement of temperature showed that at both freezing rates......, the greatest decrease in ATP content was observed when the temperature reached -0.8C. Glycolysis occurred during freezing of cod as indicated by an increase in lactate content. The changes found in all measured metabolites were more pronounced when freezing was performed at a slow rate compared to a fast rate...

Prosocial Choice in Rats Depends on Food-Seeking Behavior Displayed by Recipients.

Science.gov (United States)

Márquez, Cristina; Rennie, Scott M; Costa, Diana F; Moita, Marta A

2015-06-29

Animals often are prosocial, displaying behaviors that result in a benefit to one another [1-15] even in the absence of self-benefit [16-21] (but see [22-25]). Several factors have been proposed to modulate these behaviors, namely familiarity [6, 13, 18, 20] or display of seeking behavior [16, 21]. Rats have been recently shown to be prosocial under distress [17, 18] (but see [26-29]); however, what drives prosociality in these animals remains unclear. To address this issue, we developed a two-choice task in which prosocial behavior did not yield a benefit or a cost to the focal rat. We used a double T-maze in which only the focal rat controlled access to the food-baited arms of its own and the recipient rat's maze. In this task, the focal rat could choose between one side of the maze, which yielded food only to itself (selfish choice), and the opposite side, which yielded food to itself and the recipient rat (prosocial choice). Rats showed a high proportion of prosocial choices. By manipulating reward delivery to the recipient and its ability to display a preference for the baited arm, we found that the display of food-seeking behavior leading to reward was necessary to drive prosocial choices. In addition, we found that there was more social investigation between rats in selfish trials than in prosocial trials, which may have influenced the focals' choices. This study shows that rats provide access to food to others in the absence of added direct self-benefit, bringing new insights into the factors that drive prosociality. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

Science.gov (United States)

Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

2017-01-01

Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

Pharmacological and molecular comparison of K(ATP) channels in rat basilar and middle cerebral arteries

DEFF Research Database (Denmark)

Ploug, Kenneth Beri; Edvinsson, Lars; Olesen, Jes

2006-01-01

, we studied the possible involvement of endothelial K(ATP) channels by pressurized arteriography after luminal administration of synthetic K(ATP) channel openers to rat basilar and middle cerebral arteries. Furthermore, we examined the mRNA and protein expression profile of K(ATP) channels to rat...... basilar and middle cerebral arteries using quantitative real-time PCR (Polymerase Chain Reaction) and Western blotting, respectively. In the perfusion system, we found no significant responses after luminal application of three K(ATP) channel openers to rat basilar and middle cerebral arteries...

Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

Science.gov (United States)

Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

2015-05-01

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.

Science.gov (United States)

Vanegas, Diana C; Clark, Greg; Cannon, Ashley E; Roux, Stanley; Chaturvedi, Prachee; McLamore, Eric S

2015-12-15

The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment. Copyright © 2015 Elsevier B.V. All rights reserved.

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

Science.gov (United States)

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-12-01

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Directory of Open Access Journals (Sweden)

Ticozzi Paolo

2005-07-01

Full Text Available Abstract Background We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. Results Local, oral or intravenous administration of a single dose of oATP significantly reduced thermal hyperalgesia in hind paws of rats for 24 hours, and such effect was greater than that induced by diclofenac or indomethacin. Following oATP treatment, the expression of the pro-inflammatory chemokines interferon-gamma-inducible protein-10 (IP-10, mon ocyte chemoattractant protein-1 (MCP-1 and interleukin-8 (IL-8 within the inflamed tissues markedly decreased on vessels and infiltrated cells. In parallel, the immunohistochemical findings showed an impairment, with respect to the untreated rats, in P2X7 expression, mainly on nerves and vessels close to the site of inflammation. Finally, oATP treatment significantly reduced the presence of infiltrating inflammatory macrophages in the paw tissue. Conclusion Taken together these results clearly show that oATP reduces carrageenan-induced inflammation in rats.

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

International Nuclear Information System (INIS)

Rahman, Wira Y; Sarmini, Endang; Herlina; Lubis, Hotman; Triyanto; Hambali

2013-01-01

Adenosine triphosphate-labelled with γ- 32 P([γ- 32 p]-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- 32 P]-ATP with enzymatic reaction was used as precursors DL-glyceraldehydde 3-phosphate, Adenosine Diphosphate (ADP) and H 3 32 PO 4 , 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- 32 P] -ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia. (author)

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog.

Science.gov (United States)

Shirakawa, I; Chaen, S; Bagshaw, C R; Sugi, H

2000-02-01

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.