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Sample records for hydrolyzing released atp

  1. ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses.

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    Shuang Wu

    Full Text Available The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.

  2. 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...

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

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    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

  4. Renal epithelial cells can release ATP by vesicular fusion

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    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.

  5. 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...

  6. ATP release and purinergic signaling in NLRP3 inflammasome activation

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    Isabelle eCOUILLIN

    2013-01-01

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

  7. Cellular ATP release in the lung and airway

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    Satoru Ito

    2016-11-01

    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  8. Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

    OpenAIRE

    Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

    2006-01-01

    The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

  9. P2Y Receptor Modulation of ATP Release in the Urothelium

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    Kylie J. Mansfield

    2014-01-01

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

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

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    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.

  11. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank Bo; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout...... heart. Erythrocytes suspended in an albumin-containing saline and equilibrated at physiological Pco2 showed negligible hemolysis (... in its absence, revealing the presence of ectonucleotidase activity. The induction of either a slow (minutes) or a fast (seconds) decrease in hemoglobin O2 saturation did not lead to additional ATP release. An elevation of Pco2 was also without influence on erythrocyte ATP release. In the saline...

  12. ATP release and extracellular nucleotidase activity in erythrocytes and coronary circulation of rainbow trout

    DEFF Research Database (Denmark)

    Jensen, Frank B; Agnisola, Claudio; Novak, Ivana

    2009-01-01

    The present study tested the hypothesis that rainbow trout erythrocytes release ATP upon deoxygenation, a mechanism that enables mammalian erythrocytes to produce local vasodilation. We also investigated ATP release and ectonucleotidase activity in the coronary circulation of the isolated trout......-perfused coronary circulation, [ATP] increased as the perfusate moved through the vessels in the presence of ARL 67156. When ATP was added to the inflowing saline, most ATP disappeared during passage of the coronary bed when ARL 67156 was absent but not when it was present. We conclude that rainbow trout...

  13. ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes

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    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.

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

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    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.

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

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

    2015-01-01

    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 [3H]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. PMID:25991724

  16. ATP released by injured neurons activates Schwann cells

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    Samuele eNegro

    2016-05-01

    Full Text Available Injured nerve terminals of neuromuscular junctions (NMJs can regenerate. This remarkable and complex response is governed by molecular signals that are exchanged among the cellular components of this synapse: motor axon nerve terminal (MAT, perisynaptic Schwann cells (PSCs, and muscle fibre. The nature of signals that govern MAT regeneration is ill-known. In the present study the spider toxin α-Latrotoxin has been used as tool to investigate the mechanisms underlying peripheral neuroregeneration. Indeed this neurotoxin induces an acute, specific, localized and fully reversible damage of the presynaptic nerve terminal, and its action mimics the cascade of events that leads to nerve terminal degeneration in injured patients and in many neurodegenerative conditions. Here we provide evidence of an early release by degenerating neurons of ATP as alarm messenger, that contributes to the activation of a series of intracellular pathways within SCs that are crucial for nerve regeneration: Ca2+, cAMP, ERK1/2, and CREB. These results contribute to define the cross-talk taking place among degenerating nerve terminals and PSCs, involved in the functional recovery of the NMJ.

  17. The Contribution of Red Blood Cell Dynamics to Intrinsic Viscosity and Functional ATP Release

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    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.

  18. The Study of Alginate and Whey Protein Hydrolyzed Suplementation Utilization for Cell Release and Microencapsulated Lactobacillus Acidophilus Viability in Probiotic Ice Cream

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    Purwadi Purwadi

    2013-10-01

    Full Text Available The objectives of this research were to increase viability and activity of L. acidophilus encapsulated with alginate and whey protein hydrolyzed for cell release and microencapsulated Lactobacillus acidophilus viability in probiotic ice cream. The methods used were factorial experiment using Completely Randomized Design. Data was analysed with Variance Analysis. The results showed that the interaction between alginate and whey protein hydrolyzed supplemented could be increased the function of CaCl2 and also encapsulated L. acidophilus viability. The used alginate of 1% and whey protein hydrolyzed supplemented of 0,5% produced encapsulated L. acidophilus viability higher than before, but however, the utilization of alginate of 1% and whey protein hydrolyzed supplemented of 0% could release a few cell. Therefore, the utilization of alginate 1% and whey protein hydrolyzed supplemented 0,5% in ice cream produced L. acidophilus highest than other.   Keywords :   Lactobacillus acidophilus, microencapsulation, alginate, whey protein hydrolyzed, cell release, ice cream

  19. UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes

    DEFF Research Database (Denmark)

    Kringelbach, Tina M.; Aslan, Derya; Novak, Ivana

    2014-01-01

    the intracellular calcium concentration and that they release ATP dose-dependently upon stimulation with 1-10 μM UTP. In addition to this, osteocytes release large amounts of ATP upon cell rupture, which might also be a source for other nucleotides, such as UTP. These findings indicate that mechanically induced ATP...... the luciferin-luciferase assay and the release pathway was investigated using pharmacological inhibition. The P2Y receptor profile was analysed using gene expression analysis by reverse transcription polymerase chain reaction, while functional testing was performed using measurements of intracellular calcium...

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

    Science.gov (United States)

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

    2015-01-01

    F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

  1. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain

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    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W.; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  2. Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

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    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.

  3. ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.

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    O'Grady, Scott M; Patil, Nandadavi; Melkamu, Tamene; Maniak, Peter J; Lancto, Cheryl; Kita, Hirohito

    2013-09-15

      Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.

  4. Effect of inflammatory mediators on ATP release of human urothelial RT4 cells.

    Science.gov (United States)

    Mansfield, Kylie J; Hughes, Jessica R

    2014-01-01

    Inflammation is an important contributor to the aetiology of a number of bladder dysfunctions including interstitial cystitis, painful bladder syndrome, and overactive bladder. The aim of this study was to examine the effects of inflammatory mediators on urothelial ATP release. Human urothelial RT4 cells were exposed to normal buffer or varying concentrations of inflammatory mediators (bradykinin, histamine, and serotonin) in the presence or absence of hypotonic stretch stimuli (1 : 2 dilution of Krebs-Henseleit buffer). Others have demonstrated that bradykinin increased stretch-induced ATP release; however, we observed no change in control or stretch-induced ATP release with bradykinin. Pretreatment of RT4 cells with histamine or serotonin decreased stretch-induced ATP release (P = 0.037, P = 0.040, resp.). Previous studies have demonstrated increased ATP release in response to inflammation utilising whole bladder preparations in contrast to our simple model of cultured urothelial cells. The current study suggests that it is unlikely that there is a direct interaction between the release of inflammatory mediators and increased ATP release, but rather more complex interactions occurring in response to inflammation that lead to increased bladder sensation.

  5. ATP Release Guides Neutrophil Chemotaxis via P2Y2 and A3 Receptors

    National Research Council Canada - National Science Library

    Yu Chen; Ross Corriden; Yoshiaki Inoue; Linda Yip; Naoyuki Hashiguchi; Annelies Zinkernagel; Victor Nizet; Paul A. Insel; Wolfgang G. Junger

    2006-01-01

    .... We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors...

  6. Modulation of Central Synapses by Astrocyte-Released ATP and Postsynaptic P2X Receptors

    Science.gov (United States)

    Pankratov, Yuriy

    2017-01-01

    Communication between neuronal and glial cells is important for neural plasticity. P2X receptors are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons and/or glia. Recent data show that postsynaptic P2X receptors underlie slow neuromodulatory actions rather than fast synaptic transmission at brain synapses. Here, we review these findings with a particular focus on the release of ATP by astrocytes and the diversity of postsynaptic P2X-mediated modulation of synaptic strength and plasticity in the CNS. PMID:28845311

  7. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Novak, Ivana

    2001-01-01

    The energy providing substrate ATP can be released from various cells and act extracellularly to regulate the same cells or neighboring cells. However, the pathway for ATP release and the eliciting physiological stimulus are unclear. Recently, we showed that ATP activates P2X and P2Y purinergic...

  8. Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells

    Science.gov (United States)

    Koyama, Tetsuya; Oike, Masahiro; Ito, Yushi

    2001-01-01

    Hypotonic stress induces ATP release followed by Ca2+ oscillations in bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress-induced ATP release. Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-mediated Ca2+ oscillations induced by hypotonic stress. ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-treating the cells with Y-27632, a selective inhibitor of Rho-kinase, also suppressed the hypotonic stress-induced ATP release and Ca2+ oscillations, indicating that Rho-mediated activation of Rho-kinase may be involved in the hypotonic ATP release. Hypotonic stress also induced a transient rearrangement of the actin cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress-induced ATP release nor the Ca2+ oscillations. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathways are involved in hypotonic stress-induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs. PMID:11313444

  9. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

    Science.gov (United States)

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-06-23

    HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

  10. 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...

  11. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia

    Science.gov (United States)

    Koyanagi, Satoru; Kusunose, Naoki; Taniguchi, Marie; Akamine, Takahiro; Kanado, Yuki; Ozono, Yui; Masuda, Takahiro; Kohro, Yuta; Matsunaga, Naoya; Tsuda, Makoto; Salter, Michael W.; Inoue, Kazuhide; Ohdo, Shigehiro

    2016-01-01

    Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain. PMID:27739425

  12. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  13. Involvement of connexin 43 in ATP release from endothelial cells during reoxygenation : Role of PKA signaling pathway

    OpenAIRE

    Khawaja, Kiran

    2011-01-01

    Reperfusion-injury impairs endothelial barrier function and may lead to edema formation, impeding recovery of the reperfused heart. Recently is has been shown that ATP, spontaneously released from endothelial cells (EC) during reperfusion protects the endothelial barrier against reperfusion-injury. The aim of the present study was to identify the mechanism of this spontanous ATP release during reoxygenation in endothelia cells. The central hypothesis is that EC release ATP via a connexin-medi...

  14. Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion.

    Science.gov (United States)

    Tong, Jin; Zhou, Xiang-Dong; Perelman, Juliy M; Kolosov, Victor P

    2014-02-01

    The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA-AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT-PCR (reverse transcription-PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA-AM and RB-2, while EGTA did not have a suppressive effect. BAPTA-AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca(2+)-dependent ATP release, and nearly all Ca(2+) was provided by stored intracellular Ca(2). © 2014 The author(s).

  15. Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion

    Science.gov (United States)

    Tong, Jin; Zhou, Xiang-dong; Perelman, Juliy M.; Kolosov, Victor P.

    2013-01-01

    The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA–AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid–acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT–PCR (reverse transcription–PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA–AM and RB-2, while EGTA did not have a suppressive effect. BAPTA–AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+-dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2+. PMID:27919041

  16. 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.

  17. ATP releasing connexin 30 hemichannels mediate flow-induced calcium signaling in the collecting duct

    DEFF Research Database (Denmark)

    Svenningsen, Per; Burford, James L; Peti-Peterdi, János

    2013-01-01

    in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30(-/-) mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30...

  18. Role of Erythrocyte-released ATP in the Regulation of Microvascular Oxygen Supply in Skeletal Muscle

    Science.gov (United States)

    Ellsworth, Mary L.; Ellis, Christopher G.; Sprague, Randy S.

    2015-01-01

    In a 1914 book entitled The Respiratory Function of the Blood, Joseph Barcroft stated that “the cell takes what it needs and leaves the rest.” He postulated that there must be both a “call for oxygen” and a “mechanism by which the call elicits a response…” In the past century, intensive investigation has provided significant insights into the hemodynamic and biophysical mechanisms involved in supplying oxygen to skeletal muscle. However, the identification of the mechanism by which tissue oxygen needs are sensed and the affector responsible for altering the upstream vasculature to enable the need to be appropriately met has been a challenge. In 1995, Ellsworth et al proposed that the oxygen carrying erythrocyte, by virtue of its capacity to release the vasoactive mediator ATP in response to a decrease in oxygen saturation, could serve both roles. Several in vitro and in situ studies have established that exposure of erythrocytes to reduced oxygen tension induces the release of ATP which does result in a conducted arteriolar vasodilation with a sufficiently rapid time course to make the mechanism physiologically relevant. The components of the signaling pathway for the controlled release of ATP from erythrocytes in response to exposure to low oxygen tension have been determined. In addition, the implications of defective ATP release on human pathological conditions have been explored. This review provides a perspective on oxygen supply and the role that such a mechanism plays in meeting the oxygen needs of skeletal muscle. PMID:26336065

  19. Effects of ATP release on mucin5AC secretion in airway epithelial cells by mechanical stretching.

    Science.gov (United States)

    Zhang, Ting; Liu, Chunyi; Zhou, Xiangdong; Kolosov, Victor P; Perelman, Juliy M

    2014-01-01

    This study is to determine the effects of ATP and Ca(2+) on mucin5AC (MUC5AC) overexpression in airway epithelial cells in mechanical ventilation. Oxygen was injected into the closed box used in this study to increase the pressure. Gravity-driven draining flow led to formation of a thin liquid film on the upper portion of cell monolayer, exposing cells to the tension forces at the air-liquid interface. The levels of MUC5AC protein and ATP in culture medium were detected by ELISA and high performance liquid chromatography, respectively. Ca(2+) and MUC5AC mRNA in culture cells were detected by flow cytometry and RT-PCR, respectively. Mechanical stretching increased the expression of MUC5AC in cells and the concentration of MUC5AC and ATP in supernatant. BAPTA-AM and EGTA partially reduced the increases in the concentrations of MUC5AC and ATP in supernatant with mechanical ventilation. BAPTA-AM completely inhibited ATP in supernatant with normal breathing conditions. Our results showed that mechanical ventilation increases the secretion of MUC5AC in airway epithelial cells. This is possibly related to Ca(2+)-dependent ATP release and intracellular and external Ca(2+). © 2014 by the Association of Clinical Scientists, Inc.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid...

  1. Monitoring the hydrolyzation of aspirin during the dissolution testing for aspirin delayed-release tablets with a fiber-optic dissolution system

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2012-10-01

    Full Text Available The purpose of this study was to investigate the hydrolyzation of aspirin during the process of dissolution testing for aspirin delayed-release tablets. Hydrolysis product of salicylic acid can result in adverse effects and affect the determination of dissolution rate assaying. In this study, the technique of differential spectra was employed, which made it possible to monitor the dissolution testing in situ. The results showed that the hydrolyzation of aspirin made the percentage of salicylic acid exceed the limit of free salicylic acid (4.0, and the hydrolyzation may affect the quality detection of aspirin delayed-release tablets. Keywords: Aspirin delayed-release tablets, Drug dissolution test, Fiber-optic dissolution system, UV–vis spectrum

  2. Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus

    Science.gov (United States)

    Wall, Mark J; Dale, Nicholas

    2013-01-01

    The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources. PMID:23713028

  3. Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity.

    Science.gov (United States)

    Weitz, Jonathan R; Makhmutova, Madina; Almaça, Joana; Stertmann, Julia; Aamodt, Kristie; Brissova, Marcela; Speier, Stephan; Rodriguez-Diaz, Rayner; Caicedo, Alejandro

    2017-09-07

    Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells. To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca(2+) indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells. Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity. Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.

  4. Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia.

    Science.gov (United States)

    Zhang, X; Chen, Y; Wang, C; Huang, L-Y M

    2007-06-05

    It has been generally assumed that the cell body (soma) of a neuron, which contains the nucleus, is mainly responsible for synthesis of macromolecules and has a limited role in cell-to-cell communication. Using sniffer patch recordings, we show here that electrical stimulation of dorsal root ganglion (DRG) neurons elicits robust vesicular ATP release from their somata. The rate of release events increases with the frequency of nerve stimulation; external Ca(2+) entry is required for the release. FM1-43 photoconversion analysis further reveals that small clear vesicles participate in exocytosis. In addition, the released ATP activates P2X7 receptors in satellite cells that enwrap each DRG neuron and triggers the communication between neuronal somata and glial cells. Blocking L-type Ca(2+) channels completely eliminates the neuron-glia communication. We further show that activation of P2X7 receptors can lead to the release of tumor necrosis factor-alpha (TNFalpha) from satellite cells. TNFalpha in turn potentiates the P2X3 receptor-mediated responses and increases the excitability of DRG neurons. This study provides strong evidence that somata of DRG neurons actively release transmitters and play a crucial role in bidirectional communication between neurons and surrounding satellite glial cells. These results also suggest that, contrary to the conventional view, neuronal somata have a significant role in cell-cell signaling.

  5. TRPA1 and TRPV4 activation in human odontoblasts stimulates ATP release.

    Science.gov (United States)

    Egbuniwe, O; Grover, S; Duggal, A K; Mavroudis, A; Yazdi, M; Renton, T; Di Silvio, L; Grant, A D

    2014-09-01

    The mechanism of pain in dentine hypersensitivity is poorly understood but proposed to result from the activation of dental sensory neurons in response to dentinal fluid movements. Odontoblasts have been suggested to contribute to thermal and mechanosensation in the tooth via expression of transient receptor potential (TRP) channels. However, a mechanism by which odontoblasts could modulate neuronal activity has not been demonstrated. In this study, we investigated functional TRP channel expression in human odontoblast-like cells and measured ATP release in response to TRP channel activation. Human immortalized dental pulp cells were driven toward an odontoblast phenotype by culture in conditioned media. Functional expression of TRP channels was determined with reverse transcription polymerase chain reaction and ratiometric calcium imaging with Fura-2. ATP release was measured using a luciferin-luciferase assay. Expression of mRNA for TRPA1, TRPV1, and TRPV4 but not TRPM8 was detected in odontoblasts by reverse transcription polymerase chain reaction. Expression of TRPV4 protein was detected by Western blotting and immunocytochemistry. The TRPA1 agonists allyl isothiocyanate and cinnamaldehyde and the TRPV4 agonist GSK1016790A caused a concentration-dependent increase in intracellular Ca(2+) concentration that was inhibited by the selective antagonists HC030031, AP18, and HC067047, respectively. In contrast, exposure to the TRPV1 agonist capsaicin or the TRPM8 agonist icilin had no effect on intracellular Ca(2+) concentration. Treatment with allyl isothiocyanate, cinnamaldehyde, or GSK1016790A caused an increase in ATP concentration in culture medium that was abolished by preincubation with TRP channel antagonists. These data demonstrate that activation of TRPA1 and TRPV4 channels in human odontoblast-like cells can stimulate ATP release. We were unable to confirm the presence of thermosensitive TRPV1 and TRPM8 that has previously been reported in odontoblasts.

  6. Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular ATP levels and thiol groups

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    The experiments concerned the effect of ethacrynic acid (0.5 mM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 microM). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose...

  7. Piperine Suppresses Pyroptosis and Interleukin-1β Release upon ATP Triggering and Bacterial Infection.

    Science.gov (United States)

    Liang, Yi-Dan; Bai, Wen-Jing; Li, Chen-Guang; Xu, Li-Hui; Wei, Hong-Xia; Pan, Hao; He, Xian-Hui; Ouyang, Dong-Yun

    2016-01-01

    Piperine is a phytochemical present in black pepper (Piper nigrum Linn) and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently inhibited ATP-induced pyroptosis, thereby suppressing interleukin-1β (IL-1β) or high mobility group box-1 protein (HMGB1) release in LPS-primed bone marrow-derived macrophages and J774A.1 cells. Accompanying this, ATP-induced AMP-activated protein kinase (AMPK) activation was greatly suppressed by piperine, whereas AMPK agonist metformin counteracted piperine's inhibitory effects on pyroptosis. Moreover, piperine administration greatly reduced both peritoneal and serum IL-1β levels in the mouse model intraperitoneally infected with Escherichia coli, suggestive of suppressing systemic inflammation and pyroptosis. Our data indicated that piperine could protect macrophages from pyroptosis and reduced IL-1β and HMGB1 release by suppressing ATP-induced AMPK activation, suggesting that piperine may become a potential therapeutic agent against bacterial sepsis.

  8. Piperine suppresses pyroptosis and interleukin-1β release upon ATP triggering and bacterial infection

    Directory of Open Access Journals (Sweden)

    Yi-Dan Liang

    2016-10-01

    Full Text Available Piperine is a phytochemical present in black pepper (Piper nigrum Linn and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently inhibited ATP-induced pyroptosis, thereby suppressing interleukin-1β (IL-1β or high mobility group box-1 protein (HMGB1 release in LPS-primed bone marrow-derived macrophages (BMDMs and J774A.1 cells. Accompanying this, ATP-induced AMP-activated protein kinase (AMPK activation was greatly suppressed by piperine, whereas AMPK agonist metformin counteracted piperine’s inhibitory effects on pyroptosis. Moreover, piperine administration greatly reduced both peritoneal and serum IL-1β levels in the mouse model intraperitoneally infected with Escherichia coli, suggestive of suppressing systemic inflammation and pyroptosis. Our data indicated that piperine could protect macrophages from pyroptosis and reduced IL-1β and HMGB1 release by suppressing ATP-induced AMPK activation, suggesting that piperine may become a potential therapeutic agent against bacterial sepsis.

  9. ATP Releasing Connexin 30 Hemichannels Mediate Flow-Induced Calcium Signaling in the Collecting Duct

    Directory of Open Access Journals (Sweden)

    Per eSvenningsen

    2013-10-01

    Full Text Available ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC and AQP2. Recently, we have shown that connexin (Cx 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30-/- mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i signaling in the CD. Cortical CDs (CCDs from wild type and Cx30-/- mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30-/- CCDs ([Ca2+]i increased only 1.2-fold, p

  10. Osteoblastic Lrp4 promotes osteoclastogenesis by regulating ATP release and adenosine-A2AR signaling.

    Science.gov (United States)

    Xiong, Lei; Jung, Ji-Ung; Guo, Hao-Han; Pan, Jin-Xiu; Sun, Xiang-Dong; Mei, Lin; Xiong, Wen-Cheng

    2017-03-06

    Bone homeostasis depends on the functional balance of osteoblasts (OBs) and osteoclasts (OCs). Lrp4 is a transmembrane protein that is mutated in patients with high bone mass. Loss of Lrp4 in OB-lineage cells increases bone mass by elevating bone formation by OBs and reducing bone resorption by OCs. However, it is unclear how Lrp4 deficiency in OBs impairs osteoclastogenesis. Here, we provide evidence that loss of Lrp4 in the OB lineage stabilizes the prorenin receptor (PRR) and increases PRR/V-ATPase-driven ATP release, thereby enhancing the production of the ATP derivative adenosine. Both pharmacological and genetic inhibition of adenosine- 2A receptor (A 2A R) in culture and Lrp4 mutant mice diminishes the osteoclastogenic deficit and reduces trabecular bone mass. Furthermore, elevated adenosine-A 2A R signaling reduces receptor activator of nuclear factor κB (RANK)-mediated osteoclastogenesis. Collectively, these results identify a mechanism by which osteoblastic Lrp4 controls osteoclastogenesis, reveal a cross talk between A 2A R and RANK signaling in osteoclastogenesis, and uncover an unrecognized pathophysiological mechanism of high-bone-mass disorders. © 2017 Xiong et al.

  11. Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens

    Science.gov (United States)

    Brain, Keith L; Jackson, V Margaret; Trout, Stephen J; Cunnane, Thomas C

    2002-01-01

    A confocal Ca2+ imaging technique has been used to detect ATP release from individual sympathetic varicosities on the same nerve terminal branch. Varicose nerve terminals and smooth muscle cells in mouse vas deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA-1. Field (nerve) stimulation evoked discrete, focal increases in [Ca2+] in smooth muscle cells adjacent to identified varicosities. These focal increases in [Ca2+] have been termed ‘neuroeffector Ca2+ transients’ (NCTs). NCTs were abolished by α,β-methylene ATP (1 μM), but not by nifedipine (1 μM) or prazosin (100 nm), suggesting that NCTs are generated by Ca2+ influx through P2X receptors without a detectable contribution from L-type Ca2+ channels or α1-adrenoceptor-mediated pathways. Action potential-evoked ATP release was highly intermittent (mean probability 0.019 ± 0.002; range 0.001-0.10) at 1 Hz stimulation, even though there was no failure of action potential propagation in the nerve terminals. Twenty-eight per cent of varicosities failed to release transmitter following more than 500 stimuli. Spontaneous ATP release was very infrequent (0.0014 Hz). No Ca2+ transient attributable to noradrenaline release was detected even in response to 5 Hz stimulation. There was evidence of local noradrenaline release as the α2-adrenoceptor antagonist yohimbine increased the probability of occurrence of NCTs by 55 ± 21 % during trains of stimuli at 1 Hz. Frequency-dependent facilitation preferentially occurred at low probability release sites. The monitoring of NCTs now allows transmitter release to be detected simultaneously from each functional varicosity on an identified nerve terminal branch on an impulse-to-impulse basis. PMID:12068045

  12. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase.

    Science.gov (United States)

    Johnson, Larry; Atanasova, Kalina R; Bui, Phuong Q; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M

    2015-05-01

    Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the pro-inflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation*

    Science.gov (United States)

    Pinheiro, Ana Rita; Paramos-de-Carvalho, Diogo; Certal, Mariana; Costa, Maria Adelina; Costa, Cristina; Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Sévigny, Jean; Correia-de-Sá, Paulo

    2013-01-01

    Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors. PMID:23918924

  14. A Transient Rise in Free Mg2+Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

    Science.gov (United States)

    Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu

    2018-02-05

    For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  15. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

    Directory of Open Access Journals (Sweden)

    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  16. Real-Time Monitoring of ATP-Responsive Drug Release Using Mesoporous-Silica-Coated Multicolor Upconversion Nanoparticles.

    Science.gov (United States)

    Lai, Jinping; Shah, Birju P; Zhang, Yixiao; Yang, Letao; Lee, Ki-Bum

    2015-05-26

    Stimuli-responsive drug delivery vehicles have garnered immense interest in recent years due to unparalleled progress made in material science and nanomedicine. However, the development of stimuli-responsive devices with integrated real-time monitoring capabilities is still in its nascent stage because of the limitations of imaging modalities. In this paper, we describe the development of a polypeptide-wrapped mesoporous-silica-coated multicolor upconversion nanoparticle (UCNP@MSN) as an adenosine triphosphate (ATP)-responsive drug delivery system (DDS) for long-term tracking and real-time monitoring of drug release. Our UCNP@MSN with multiple emission peaks in UV-NIR wavelength range was functionalized with zinc-dipicolylamine analogue (TDPA-Zn(2+)) on its exterior surface and loaded with small-molecule drugs like chemotherapeutics in interior mesopores. The drugs remained entrapped within the UCNP-MSNs when the nanoparticles were wrapped with a compact branched polypeptide, poly(Asp-Lys)-b-Asp, because of multivalent interactions between Asp moieties present in the polypeptide and the TDPA-Zn(2+) complex present on the surface of UCNP-MSNs. This led to luminescence resonance energy transfer (LRET) from the UCNPs to the entrapped drugs, which typically have absorption in UV-visible range, ultimately resulting in quenching of UCNP emission in UV-visible range while retaining their strong NIR emission. Addition of ATP led to a competitive displacement of the surface bound polypeptide by ATP due to its higher affinity to TDPA-Zn(2+), which led to the release of the entrapped drugs and subsequent elimination of LRET. Monitoring of such ATP-triggered ratiometric changes in LRET allowed us to monitor the release of the entrapped drugs in real-time. Given these results, we envision that our proposed UCNP@MSN-polypeptide hybrid nanoparticle has great potential for stimuli-responsive drug delivery as well as for monitoring biochemical changes taking place in live cancer

  17. Molecular devices for the regulation of chloroplast ATP synthase

    NARCIS (Netherlands)

    Hisabori, T.; Konno, H.; Ichimura, H.; Strotmann, H.; Bald, D.

    2002-01-01

    In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential

  18. Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Haanes, Kristian Agmund; Christensen, Nynne

    2015-01-01

    BACKGROUND: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic si...

  19. Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome.

    Science.gov (United States)

    Rodella, Umberto; Negro, Samuele; Scorzeto, Michele; Bergamin, Elisanna; Jalink, Kees; Montecucco, Cesare; Yuki, Nobuhiro; Rigoni, Michela

    2017-05-01

    The neuromuscular junction is exposed to different types of insult, including mechanical trauma, toxins and autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain-Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, which activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regeneration program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron-derived ATP acts as an alarm messenger for Schwann cells, where it induces the activation of intracellular pathways, including calcium signaling, cAMP and CREB, which, in turn, produce signals that promote nerve regeneration. These results contribute to defining the cross-talk taking place at the neuromuscular junction when it is attacked by anti-gangliosides autoantibodies plus complement, which is crucial for nerve regeneration and is also likely to be important in other peripheral neuropathies. © 2017. Published by The Company of Biologists Ltd.

  20. Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome

    Directory of Open Access Journals (Sweden)

    Umberto Rodella

    2017-05-01

    Full Text Available The neuromuscular junction is exposed to different types of insult, including mechanical trauma, toxins and autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain–Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, which activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regeneration program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron-derived ATP acts as an alarm messenger for Schwann cells, where it induces the activation of intracellular pathways, including calcium signaling, cAMP and CREB, which, in turn, produce signals that promote nerve regeneration. These results contribute to defining the cross-talk taking place at the neuromuscular junction when it is attacked by anti-gangliosides autoantibodies plus complement, which is crucial for nerve regeneration and is also likely to be important in other peripheral neuropathies.

  1. Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels.

    Science.gov (United States)

    Price, Alexander K; Fischer, David J; Martin, R Scott; Spence, Dana M

    2004-08-15

    The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 micron (2.22 +/- 0.50 microM ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 +/- 0.15 microM) vs a microchip (2.59 +/- 0.32 microM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

  2. Disruption of lolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal for Escherichia coli and Prevents Release of Lipoproteins from the Inner Membrane

    OpenAIRE

    Narita, Shin-ichiro; Tanaka, Kimie; Matsuyama, Shin-ichi; Tokuda, Hajime

    2002-01-01

    ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstituti...

  3. Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C.

    Science.gov (United States)

    Weng, Ju-Yun; Hsu, Tsan-Ting; Sun, Synthia H

    2008-05-15

    A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes. Copyright 2007 Wiley-Liss, Inc.

  4. Evidence against ATP being the inhibitory transmitter released by nonadrenergic noncholinergic nerves in the canine ileocolonic junction

    NARCIS (Netherlands)

    Boeckxstaens, G. E.; Pelckmans, P. A.; Rampart, M.; Verbeuren, T. J.; Herman, A. G.; van Maercke, Y. M.

    1990-01-01

    The nonadrenergic noncholinergic (NANC) relaxations in response to electrical stimulation and acetylcholine in the canine terminal ileum and ileocolonic junction were further characterized and the possible involvement of the putative NANC neurotransmitter ATP was investigated. During a

  5. Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation*

    Science.gov (United States)

    Weihs, Anna M.; Fuchs, Christiane; Teuschl, Andreas H.; Hartinger, Joachim; Slezak, Paul; Mittermayr, Rainer; Redl, Heinz; Junger, Wolfgang G.; Sitte, Harald H.; Rünzler, Dominik

    2014-01-01

    Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing. PMID:25118288

  6. Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

    Science.gov (United States)

    Weihs, Anna M; Fuchs, Christiane; Teuschl, Andreas H; Hartinger, Joachim; Slezak, Paul; Mittermayr, Rainer; Redl, Heinz; Junger, Wolfgang G; Sitte, Harald H; Rünzler, Dominik

    2014-09-26

    Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Prevention of anti-microbial peptide LL-37-induced apoptosis and ATP release in the urinary bladder by a modified glycosaminoglycan.

    Directory of Open Access Journals (Sweden)

    Won Yong Lee

    Full Text Available Interstitial cystitis (IC, often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1 the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2 the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3 the physiological and molecular markers that correlate with the severity of the inflammation, and (4 the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.

  8. Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts.

    Science.gov (United States)

    Hu, Fen; Hui, Zhenhai; Wei, Wei; Yang, Jianyu; Chen, Ziyuan; Guo, Bu; Xing, Fulin; Zhang, Xinzheng; Pan, Leiting; Xu, Jingjun

    2017-04-22

    Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca 2+ ] c ) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects were almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca 2+ channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca 2+ entry through activation of TRPV4 channel in synoviocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. The antiviral drug tenofovir, an inhibitor of Pannexin-1-mediated ATP release, prevents liver and skin fibrosis by downregulating adenosine levels in the liver and skin.

    Directory of Open Access Journals (Sweden)

    Jessica L Feig

    Full Text Available Fibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis.Thioacetamide (100mg/kg IP-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ (n = 5-10. Bleomycin (0.25U, SubQ-treated mice were treated with vehicle or tenofovir (75mg/kg, IP (n = 5-10. Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA.Treatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1.These studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.

  10. Association of the α2δ1 Subunit with Cav3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

    Science.gov (United States)

    Thompson, William R.; Majid, Amber S.; Czymmek, Kirk J.; Ruff, Albert L.; García, Jesús; Duncan, Randall L.; Farach-Carson, Mary C.

    2015-01-01

    Voltage sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α1 pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs, but a relatively small amount of L-type α1 subunits. Auxiliary VSCC subunits have several functions including modulating gating kinetics, trafficking of the channel and phosphorylation events. The influence of the α2δ auxiliary subunit on T-type VSCCs and the physiological consequences of that association are incompletely understood and have yet to be investigated in bone. In this study, we postulated that the auxiliary α2δ subunit of the VSCC complex modulates mechanically-regulated ATP release in osteocytes via its association with the T-type, Cav3.2 (α1H) subunit. We demonstrated by RT-PCR, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type, Cav3.2 (α1H) subunit more abundantly than the L-type, Cav1.2 (α1C). We also demonstrated that the α2δ1 subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Cav3.2 (α1H) subunit in MLO-Y4 cells. Interestingly, siRNA mediated knockdown of α2δ1 completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α2δ1 subunit and resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α2δ1 knockdown showed decreased membrane localization of Cav3.2 (α1H) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Cav3.2 (α1H) at the cell membrane. PMID:21638318

  11. Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production.

    Science.gov (United States)

    Krick, Stefanie; Wang, Junjie; St-Pierre, Melissa; Gonzalez, Carlos; Dahl, Gerhard; Salathe, Matthias

    2016-03-18

    Human airway epithelial cells express pannexin 1 (Panx1) channels to release ATP, which regulates mucociliary clearance. Airway inflammation causes mucociliary dysfunction. Exposure of primary human airway epithelial cell cultures to IFN-γ for 48 h did not alter Panx1 protein expression but significantly decreased ATP release in response to hypotonic stress. The IFN-γ-induced functional down-regulation of Panx1 was due to the up-regulation of dual oxidase 2 (Duox2). Duox2 suppression by siRNA led to an increase in ATP release in control cells and restoration of ATP release in cells treated with IFN-γ. Both effects were reduced by the pannexin inhibitor probenecid. Duox2 up-regulation stoichiometrically increases H2O2 and proton production. H2O2 inhibited Panx1 function temporarily by formation of disulfide bonds at the thiol group of its terminal cysteine. Long-term exposure to H2O2, however, had no inhibitory effect. To assess the role of cellular acidification upon IFN-γ treatment, fully differentiated airway epithelial cells were exposed to ammonium chloride to alkalinize the cytosol. This led to a 2-fold increase in ATP release in cells treated with IFN-γ that was also inhibited by probenecid. Duox2 knockdown also partially corrected IFN-γ-mediated acidification. The direct correlation between intracellular pH and Panx1 open probability was shown in oocytes. Therefore, airway epithelial cells release less ATP in response to hypotonic stress in an inflammatory environment (IFN-γ exposure). Decreased Panx1 function is a response to cell acidification mediated by IFN-γ-induced up-regulation of Duox2, representing a novel mechanism for mucociliary dysfunction in inflammatory airway diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Mechanosensory and ATP Release Deficits following Keratin14-Cre-Mediated TRPA1 Deletion Despite Absence of TRPA1 in Murine Keratinocytes.

    Directory of Open Access Journals (Sweden)

    Katherine J Zappia

    Full Text Available Keratinocytes are the first cells that come into direct contact with external tactile stimuli; however, their role in touch transduction in vivo is not clear. The ion channel Transient Receptor Potential Ankyrin 1 (TRPA1 is essential for some mechanically-gated currents in sensory neurons, amplifies mechanical responses after inflammation, and has been reported to be expressed in human and mouse skin. Other reports have not detected Trpa1 mRNA transcripts in human or mouse epidermis. Therefore, we set out to determine whether selective deletion of Trpa1 from keratinocytes would impact mechanosensation. We generated K14Cre-Trpa1fl/fl mice lacking TRPA1 in K14-expressing cells, including keratinocytes. Surprisingly, Trpa1 transcripts were very poorly detected in epidermis of these mice or in controls, and detection was minimal enough to preclude observation of Trpa1 mRNA knockdown in the K14Cre-Trpa1fl/fl mice. Unexpectedly, these K14Cre-Trpa1fl/fl mice nonetheless exhibited a pronounced deficit in mechanosensitivity at the behavioral and primary afferent levels, and decreased mechanically-evoked ATP release from skin. Overall, while these data suggest that the intended targeted deletion of Trpa1 from keratin 14-expressing cells of the epidermis induces functional deficits in mechanotransduction and ATP release, these deficits are in fact likely due to factors other than reduction of Trpa1 expression in adult mouse keratinocytes because they express very little, if any, Trpa1.

  13. Mechanism of ATP turnover inhibition in the EJC

    Science.gov (United States)

    Nielsen, Klaus H.; Chamieh, Hala; Andersen, Christian B.F.; Fredslund, Folmer; Hamborg, Kristiane; Le Hir, Hervé; Andersen, Gregers R.

    2009-01-01

    The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF3, the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC–ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5–RNA–ADP–AlF3, suggesting that the use of ADP–AlF3 may be a valuable tool for examining DEAD-box ATPases in general. PMID:19033377

  14. The food dye FD&C Blue No. 1 is a selective inhibitor of the ATP release channel Panx1.

    Science.gov (United States)

    Wang, Junjie; Jackson, David George; Dahl, Gerhard

    2013-05-01

    The food dye FD&C Blue No. 1 (Brilliant Blue FCF [BB FCF]) is structurally similar to the purinergic receptor antagonist Brilliant Blue G (BBG), which is a well-known inhibitor of the ionotropic P2X7 receptor (P2X7R). The P2X7R functionally interacts with the membrane channel protein pannexin 1 (Panx1) in inflammasome signaling. Intriguingly, ligands to the P2X7R, regardless of whether they are acting as agonists or antagonists at the receptor, inhibit Panx1 channels. Thus, because both P2X7R and Panx1 are inhibited by BBG, the diagnostic value of the drug is limited. Here, we show that the food dye BB FCF is a selective inhibitor of Panx1 channels, with an IC50 of 0.27 µM. No significant effect was observed with concentrations as high as 100 µM of BB FCF on P2X7R. Differing by just one hydroxyl group from BB FCF, the food dye FD&C Green No. 3 exhibited similar selective inhibition of Panx1 channels. A reverse selectivity was observed for the P2X7R antagonist, oxidized ATP, which in contrast to other P2X7R antagonists had no significant inhibitory effect on Panx1 channels. Based on its selective action, BB FCF can be added to the repertoire of drugs to study the physiology of Panx1 channels. Furthermore, because Panx1 channels appear to be involved directly or indirectly through P2X7Rs in several disorders, BB FCF and derivatives of this "safe" food dye should be given serious consideration for pharmacological intervention of conditions such as acute Crohn's disease, stroke, and injuries to the central nervous system.

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

    Science.gov (United States)

    Gregory, Nicholas S; Whitley, Phillip E; Sluka, Kathleen A

    2015-01-01

    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.

  16. 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.

  17. Astrocytic Lrp4 (Low-Density Lipoprotein Receptor-Related Protein 4) Contributes to Ischemia-Induced Brain Injury by Regulating ATP Release and Adenosine-A2AR (Adenosine A2A Receptor) Signaling.

    Science.gov (United States)

    Ye, Xin-Chun; Hu, Jin-Xia; Li, Lei; Li, Qiang; Tang, Fu-Lei; Lin, Sen; Sun, Dong; Sun, Xiang-Dong; Cui, Gui-Yun; Mei, Lin; Xiong, Wen-Cheng

    2018-01-01

    Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. The brain-specific Lrp4 conditional knockout mice (Lrp4 GFAP-Cre ), astrocytic-specific Lrp4 conditional knockout mice (Lrp4 GFAP-creER ), and their control mice (Lrp4 f/f ) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A 2A R (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4 GFAP-Cre and Lrp4 GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P 2 X 7 R or adenosine-A 2A R signaling diminished Lrp4 GFAP-creER 's protective effect. The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A 2A R signaling. © 2017 American Heart

  18. Voltage Dependence of ATP Secretion in Mammalian Taste Cells

    Science.gov (United States)

    Romanov, Roman A.; Rogachevskaja, Olga A.; Khokhlov, Alexander A.; Kolesnikov, Stanislav S.

    2008-01-01

    Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca2+ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels. PMID:19029378

  19. Hydrolysis of ATP at only one GyrB subunit is sufficient to promote supercoiling by DNA gyrase

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1998-01-01

    Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed, and the ability of such complexes to hydrolyze ATP was investigated. We found that ATP hydrolysis...

  20. 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

  1. Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

    Science.gov (United States)

    Tuena de Gómez-Puyou, M; Pérez-Hernández, G; Gómez-Puyou, A

    1999-12-01

    increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.

  2. 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......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...... mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream...

  3. Effect of ATP depletion and temperature on the transferrin-mediated uptake and release of iron by BeWo choriocarcinoma cells

    NARCIS (Netherlands)

    van der Ende, A.; du Maine, A.; Schwartz, A. L.; Strous, G. J.

    1989-01-01

    We have recently described the transferrin-mediated uptake and release of iron by BeWo cells [van der Ende, du Maine, Simmons, Schwartz & Strous (1987) J. Biol. Chem. 262, 8910-8916]. We now extend our studies of the mechanisms responsible for uptake and release of iron by these cells. Following

  4. Association of the α(2)δ(1) subunit with Ca(v)3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes.

    Science.gov (United States)

    Thompson, William R; Majid, Amber S; Czymmek, Kirk J; Ruff, Albert L; García, Jesús; Duncan, Randall L; Farach-Carson, Mary C

    2011-09-01

    Voltage-sensitive calcium channels (VSCCs) mediate signaling events in bone cells in response to mechanical loading. Osteoblasts predominantly express L-type VSCCs composed of the α(1) pore-forming subunit and several auxiliary subunits. Osteocytes, in contrast, express T-type VSCCs and a relatively small amount of L-type α(1) subunits. Auxiliary VSCC subunits have several functions, including modulating gating kinetics, trafficking of the channel, and phosphorylation events. The influence of the α(2)δ auxiliary subunit on T-type VSCCs and the physiologic consequences of that association are incompletely understood and have yet to be investigated in bone. In this study we postulated that the auxiliary α(2) δ subunit of the VSCC complex modulates mechanically regulated ATP release in osteocytes via its association with the T-type Ca(v) 3.2 (α(1H) ) subunit. We demonstrated by reverse-transcriptase polymerase chain reaction, Western blotting, and immunostaining that MLO-Y4 osteocyte-like cells express the T-type Ca(v)3.2(α(1H)) subunit more abundantly than the L-type Ca(v)1.2 (α(1C)) subunit. We also demonstrated that the α(2) δ(1) subunit, previously described as an L-type auxiliary subunit, complexes with the T-type Ca(v)3.2 (α(1H)) subunit in MLO-Y4 cells. Interestingly, siRNA-mediated knockdown of α(2) δ(1) completely abrogated ATP release in response to membrane stretch in MLO-Y4 cells. Additionally, knockdown of the α(2)δ(1) subunit resulted in reduced ERK1/2 activation. Together these data demonstrate a functional VSCC complex. Immunocytochemistry following α(2)δ(1) knockdown showed decreased membrane localization of Ca(v) 3.2 (α(1H)) at the plasma membrane, suggesting that the diminished ATP release and ERK1/2 activation in response to membrane stretch resulted from a lack of Ca(v) 3.2 (α(1H)) at the cell membrane. Copyright © 2011 American Society for Bone and Mineral Research.

  5. Hydrolyzable tannin analysis in food.

    Science.gov (United States)

    Arapitsas, Panagiotis

    2012-12-01

    The discovery of plant polyphenols in food is perhaps one of the biggest breakthroughs in modern food science. Plant polyphenols are known for their role in food quality and safety, since they contribute significantly to taste, flavour, colour, stability etc., while they are increasingly recognised as important factors in long-term health, contributing towards reducing the risk of chronic disease. Almost 200years ago, hydrolyzable tannins (HTs) were the first group of plant polyphenols subjected to analytical chemical research. Despite the lack of commercially available standards, food analysis research offers a wealth of papers dealing with extraction optimisation, identification and quantification of HTs. The object of this review is to summarise analytical chemistry applications and the tools currently used for the analysis of HTs in food. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. 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

  7. Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

    Science.gov (United States)

    Denis, María Florencia Leal; Incicco, J. Jeremías; Espelt, María Victoria; Verstraeten, Sandra V.; Pignataro, Omar P.; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2014-01-01

    SUMMARY Background The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). Methods Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. Results Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. Conclusions MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General Significance Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation. PMID:23742824

  8. Marine biofouling resistance of polyurethane with biodegradation and hydrolyzation.

    Science.gov (United States)

    Xu, Wentao; Ma, Chunfeng; Ma, Jielin; Gan, Tiansheng; Zhang, Guangzhao

    2014-03-26

    We have prepared polyurethane with poly(ε-caprolactone) (PCL) as the segments of the main chain and poly(triisopropylsilyl acrylate) (PTIPSA) as the side chains by a combination of radical polymerization and a condensation reaction. Quartz crystal microbalance with dissipation studies show that polyurethane can degrade in the presence of enzyme and the degradation rate decreases with the PTIPSA content. Our studies also demonstrate that polyurethane is able to hydrolyze in artificial seawater and the hydrolysis rate increases as the PTIPSA content increases. Moreover, hydrolysis leads to a hydrophilic surface that is favorable to reduction of the frictional drag under dynamic conditions. Marine field tests reveal that polyurethane has good antifouling ability because polyurethane with a biodegradable PCL main chain and hydrolyzable PTIPSA side chains can form a self-renewal surface. Polyurethane was also used to carry and release a relatively environmentally friendly antifoulant, and the combined system exhibits a much higher antifouling performance even in a static marine environment.

  9. Hydrolyzable tannins from Balanophora polyandra

    Directory of Open Access Journals (Sweden)

    Yangai Wang

    2013-02-01

    Full Text Available This study reports an investigation of the chemical constituents of Balanophora polyandra Griff. Fifteen compounds were isolated by column chromatography on silica gel, Toyo-pearl HW-40C, Sephadex LH-20 and by HPLC. Their structures were elucidated as 1,4-di-O-galloyl-2-O-[(E-p-coumaroyl]-β-D-glucopyranose (1, 1-O-galloyl-β-D-pyranglucose (2, 1-p-coumaryl-β-D-pyranglucose (3, 1-O-(E-caffeoyl-β-D-pyranglucose (4, 1,3-di-O-galloyl-β-D-pyranglucose (5, 1,6-di-O-galloyl-β-D-pyranglucose (6, 1-O-(E-caffeoyl-4-O-galloyl-β-D-pyranglucose (7, 1-O-(E-caffeoyl-6-O-galloyl-β-D-pyranglucose (8, 1-O-(E-caffeoyl-4,6-di-O-galloyl-β-D-pyranglucose (9, 1-O-(E-caffeoyl-4,6-(S-HHDP-β-D-pyranglucose (10, 1,2,3,6-tetra-O-galloyl-β-D-pyranglucose (11, 4,6-(S-hexahydroxydiphenoyl-(α/β-D-glucose (12, 1-O-(E-caffeoyl-4,6-[1′,1″-(3′,3″,4′,4″-tetrahydroxydibenzofurandicarboxyl]-β-D-glucopyranose (13, flavogallonic acid (14, and phloretin-4′-O-β-D-glucoside (15 on the basis of spectral analysis. Compound 1 was a new hydrolyzable tannin, 9 was obtained from this genus for the first time, and compounds 5, 6 and 11–14 were isolated from this plant for the first time.

  10. Hydrolyzed Vegetable Protein Containing Products Recalls

    Data.gov (United States)

    U.S. Department of Health & Human Services — This list includes products subject to recall in the United States since February 2010 related to hydrolyzed vegetable protein (HVP) paste and powder distributed by...

  11. Hydrolyzable polyureas bearing hindered urea bonds.

    Science.gov (United States)

    Ying, Hanze; Cheng, Jianjun

    2014-12-10

    Hydrolyzable polymers are widely used materials that have found numerous applications in biomedical, agricultural, plastic, and packaging industrials. They usually contain ester and other hydrolyzable bonds, such as anhydride, acetal, ketal, or imine, in their backbone structures. Here, we report the first design of hydrolyzable polyureas bearing dynamic hindered urea bonds (HUBs) that can reversibly dissociate to bulky amines and isocyanates, the latter of which can be further hydrolyzed by water, driving the equilibrium to facilitate the degradation of polyureas. Polyureas bearing 1-tert-butyl-1-ethylurea bonds that show high dynamicity (high bond dissociation rate), in the form of either linear polymers or cross-linked gels, can be completely degraded by water under mild conditions. Given the simplicity and low cost for the production of polyureas by simply mixing multifunctional bulky amines and isocyanates, the versatility of the structures, and the tunability of the degradation profiles of HUB-bearing polyureas, these materials are potentially of very broad applications.

  12. 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

  13. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

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

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a dire...... shedding, possibly affecting the structural plasticity associated with learning and memory.......Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a direct...... ATP-NCAM interaction and discuss its functional implications. The ectodomain of NCAM contains the ATP binding Walker motif A and has intrinsic ATPase activity, which could modulate NCAM-dependent signaling processes. NCAM interacts directly with and signals through FGFR. The NCAM binding site to ATP...

  15. Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

    Science.gov (United States)

    Ding, Yi; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation. PMID:20826566

  16. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction

    DEFF Research Database (Denmark)

    Kringelbach, Tina M.; Aslan, Derya; Novak, Ivana

    2015-01-01

    effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP...

  17. 21 CFR 573.540 - Hydrolyzed leather meal.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Hydrolyzed leather meal. 573.540 Section 573.540... Additive Listing § 573.540 Hydrolyzed leather meal. (a) Identity. Hydrolyzed leather meal is produced from leather scraps that are treated with steam for not less than 33 minutes at a pressure of not less than 125...

  18. Hydrolyzable tannins in Bixa Orellana L.

    OpenAIRE

    Ricardo Jorge Cruz Lima; Antonio Jeferson de Deus Moreno; Solange Fernanda Loureiro de Castro; José de Ribamar Santos Gonçalves; Antonio Benedito de Olivera; José Marcos Sasaki; Paulo de Tarso Cavalcante Freire

    2006-01-01

    The aqueous material found in the fruits of Bixa Orellana L. was collected, dried, and characterized using several experimental techniques, namely phytochemical analysis in order to identify the biologically active constituents, Fourier transform infrared (FT-IR) spectroscopy for vibrational analysis, and X-ray powder diffraction in order to identify the presence of crystalline phases in the sample. The results showed that the aqueous material possesses high concentrations of hydrolyzable tan...

  19. Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor.

    Science.gov (United States)

    Ozalp, Veli C; Pedersen, Tina R; Nielsen, Lise J; Olsen, Lars F

    2010-11-26

    Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

  20. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

    Directory of Open Access Journals (Sweden)

    M Florencia Leal Denis

    Full Text Available The peptide mastoparan 7 (MST7 triggered in human erythrocytes (rbcs the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe, interacting with P (purinergic receptors, can affect cell volume (Vr, we explored the dynamic regulation between Vr and ATPe.We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors.In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%.Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

  1. Structure and mechanism of ATP-dependent phospholipid transporters

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Poulsen, Lisbeth Rosager; Bailly, Aurélien

    2015-01-01

    , 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......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...

  2. Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP.

    Science.gov (United States)

    Martín-Estebané, María; Navascués, Julio; Sierra-Martín, Ana; Martín-Guerrero, Sandra M; Cuadros, Miguel A; Carrasco, María-Carmen; Marín-Teva, José L

    2017-01-01

    Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7th day of incubation (E7), subsequently colonizing the entire retina by central-to-peripheral tangential migration, as previously shown by our group. The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the

  3. Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

    Science.gov (United States)

    Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

    2013-06-01

    A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

  4. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    Science.gov (United States)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

  5. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism.

    Science.gov (United States)

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2015-08-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd(3+) caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd(3+). Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd(3+), while NO donors rescued apyrase- and Gd(3+)-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd(3+)-sensitive receptor that is coupled with intracellular NO production. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue.

    Science.gov (United States)

    Balaz, Martina; Sundberg, Mark; Persson, Malin; Kvassman, Jan; Månsson, Alf

    2007-06-19

    Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.

  7. Selection and production of insoluble xylan hydrolyzing enzyme by ...

    African Journals Online (AJOL)

    Forty-two strains of Thermomyces lanuginosus isolated from various sources in Thailand were divide into 4 groups based on the soluble xylan hydrolyzing (SXH) and insoluble xylan hydrolyzing (IXH) enzyme activities in the supernatant obtained from 5-day culture at 50°C in the liquid medium using corncob as substrate.

  8. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants

    OpenAIRE

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    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, includ...

  9. Chemical and proteolysis-derived changes during long-term storage of lactose-hydrolyzed ultrahigh-temperature (UHT) milk.

    Science.gov (United States)

    Jansson, Therese; Jensen, Hanne B; Sundekilde, Ulrik K; Clausen, Morten R; Eggers, Nina; Larsen, Lotte B; Ray, Colin; Andersen, Henrik J; Bertram, Hanne C

    2014-11-19

    Proteolytic activity in milk may release bitter-tasting peptides and generate free amino terminals that react with carbohydrates, which initiate Maillard reaction. Ultrahigh temperature (UHT) heat treatment inactivates the majority of proteolytic enzymes in milk. In lactose-hydrolyzed milk a β-galactosidase preparation is applied to the milk after heat treatment, which has proteolytic side activities that may induce quality deterioration of long-term-stored milk. In the present study proteolysis, glycation, and volatile compound formation were investigated in conventional (100% lactose), filtered (60% lactose), and lactose-hydrolyzed (UHT milk using reverse phase high-pressure liquid chromatography-mass spectrometry, proton nuclear magnetic resonance, and gas chromatography-mass spectrometry. Proteolysis was observed in all milk types. However, the degree of proteolysis was significantly higher in the lactose-hydrolyzed milk compared to the conventional and filtered milk. The proteins most prone to proteolysis were β-CN and αs1-CN, which were clearly hydrolyzed after approximately 90 days of storage in the lactose-hydrolyzed milk.

  10. 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.

  11. Towards the development of an automated ATP measuring platform to monitor microbial quality of drinking water

    DEFF Research Database (Denmark)

    Tatari, Karolina; Hansen, C. B.; Rasmussen, A.

    This work aimed to develop an automated and nearly on-line method to monitor ATP levels in drinking water as an indicator of microbial contamination. The system consists of a microfluidic cartridge installed in a light tight box, where the sample is mixed with the reagents and the emitted light...... is detected by a photomultiplier. Temperature in the assay box is controlled and set to 25°C. Calibration of the system using ATP standard solutions was successful, both for free and for total ATP. Chemical release of ATP by reagent addition however resulted in the formation of particles that ultimately...... clogged the microfluidic channels. An alternative thermal lysis step was implemented, by adding a flow-though heating/cooling step to the system. Thermal lysis showed efficient release of ATP from an E. coli dilution, but the releasing efficiency varied according to the type of water. Overall...

  12. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants1[OPEN

    Science.gov (United States)

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-01-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. PMID:26224801

  13. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants.

    Science.gov (United States)

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-09-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg(2+) as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. © 2015 American Society of Plant Biologists. All Rights Reserved.

  14. Removal of inhibitors from lignocellulosic hydrolyzates by vacuum membrane distillation.

    Science.gov (United States)

    Chen, Jingwen; Zhang, Yaqin; Wang, Yafei; Ji, Xiaosheng; Zhang, Lin; Mi, Xigeng; Huang, He

    2013-09-01

    In this study, vacuum membrane distillation (VMD) was used to remove two prototypical fermentation inhibitors (acetic acid and furfural) from lignocellulose hydrolyzates. The effect of operating parameters, such as feed temperature and feed velocity, on the removal efficiencies of inhibitors was investigated. Under optimal conditions, more than 98% of furfural could be removed by VMD. However, the removal efficiency of acetic acid was considerably lower. After furfural and acetic acid were selectively removed from hydrolyzates by VMD, ethanol production efficiency increased by 17.8% compared to original hydrolyzates. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. 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.

  16. Combustion of Pure, Hydrolyzed and Methyl Ester Formed of Jatropha Curcas Lin oil

    Directory of Open Access Journals (Sweden)

    Muhaji Muhaji

    2015-10-01

    Full Text Available The density and viscosity of vegetable oil are higher than that of diesel oil. Thus its direct combustion in the diesel engine results many problems. This research was conducted to investigate the flame characteristics of combustion of jatropha curcas lin in pure, hydrolyzed and methyl ester form. The results indicated that the combustion of pure jatropha curcas lin occurs in three stages, hydrolyzed in two stages    and methyl ester in one stage. For pure jatropha curcas lin, in the first stage, unsaturated fatty acid burned for  0.265 s.  It is followed by saturated fatty acid, burned for 0.389 s in the second stage. And, in the last stage is the burned of glycerol for 0.560 s. Meanwhile for hydrolyzed one, in the first stage, unsaturated fatty acid burned for 0.736 s, followed by saturated fatty acid, burned  for 0.326 s in the second stage. And the last, for methyl ester is the burned for 0.712 s. The highest burning rate was for methyl ester which was 0.003931cc/s. The energy releasing rate of methyl ester, which was for 13,628.67 kcal/(kg.s resembled that of diesel oil the most, while the lowest rate was for pure jatropha curcas lin which was 8,200.94 kcal/(kg.s. In addition, massive explosion occurred in the fuel containing unsaturated fatty acid and glycerol

  17. Mode of operation in fermentation of dilute acid hydrolyzates

    Energy Technology Data Exchange (ETDEWEB)

    Liden, Gunnar [Chalmers Univ. of Technology, Goeteborg (Sweden). Dept. of Chemical Reaction Engineering; Gustafsson, Lena [Goeteborg Univ. (Sweden). Dept. of General and Marine Microbiology

    2002-05-01

    This report describes work done to find process solution for fermentation of dilute acid hydrolyzates. The work was carried out in the period July 1997 - December 1998. Financial support was initially given by the Swedish National Board of Technical Development, but as of July 1998 support was instead given by the Swedish National Energy Agency. The main findings and achievements of the project are summarized below: 1. It has been found possible to ferment strongly inhibiting hydrolyzates from both spruce and birch using fed-batch technique, i.e. by slowly feeding hydrolyzate to the fermentor. 2. A very promising on-line control algorithm for use in fed-batch fermentation, even with varying hydrolyzate composition, has been developed and tested for a range of different substrates. Previously unreported metabolites, of possible importance in the acute inhibition of glycolysis, have been identified in studies of kinetics of conversion of the inhibitors furfural and HMF in model synthetic media.

  18. Mode of operation in fermentation of dilute acid hydrolyzates

    Energy Technology Data Exchange (ETDEWEB)

    Liden, Gunnar [Chalmers Univ. of Technology, Goeteborg (Sweden). Dept. of Chemical Reaction Engineering; Gustafsson, Lena [Goeteborg Univ. (Sweden). Dept. of General and Marine Microbiology

    2000-06-01

    This report describes work done to find process solution for fermentation of dilute acid hydrolyzates. The work was carried out in the period July 1997 - December 1998. Financial support was initially given by the Swedish National Board of Technical Development, but as of July 1998 support was instead given by the Swedish National Energy Administration. The main findings and achievements of the project are summarized below: 1. It has been found possible to ferment strongly inhibiting hydrolyzates from both spruce and birch using fed-batch technique, i.e. by slowly feeding hydrolyzate to the fermentor. 2. A very promising on-line control algorithm for use in fed-batch fermentation, even with varying hydrolyzate composition, has been developed and tested for a range of different substrates. 3. Previously unreported metabolites, of possible importance in the acute inhibition of glycolysis, have been identified in studies of kinetics of conversion of the inhibitors furfural and HMF in model synthetic media.

  19. Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP).

    Science.gov (United States)

    Little, J S; Broomfield, C A; Fox-Talbot, M K; Boucher, L J; MacIver, B; Lenz, D E

    1989-01-01

    The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.

  20. Hydrolyzed polyacrylamide grafted carboxymethylxyloglucan based microbeads for pH responsive drug delivery.

    Science.gov (United States)

    Setty, C Mallikarjuna; Deshmukh, Anand S; Badiger, Aravind M

    2014-06-01

    The present study investigates the pharmaceutical application of hydrolyzed polyacrylamide grafted carboxymethylxyloglucan (HPam-g-CMXG), as promising polymeric material for the development of pH responsive microbeads. The graft copolymer was synthesized by conventional free radical polymerization method and saponified to enhance its functionality and characterized. An acute oral toxicity study ensured the bio-safety of developed copolymer for clinical application. Various batches of pH responsive spherical microbeads were developed and evaluated for the effect of process parameters on their overall performance. Result of in vitro drug release study (USP Type-II, paddle method) carried out in two different pH media (pH 1.2 and pH 7.4) showed a triphasic drug release pattern in all the formulations. Both the drug release and swelling of microbeads were significantly higher in simulated intestinal (alkaline) pH compared to simulated gastric (acidic) pH and this nature is desirable for targeted drug delivery. A strong correlation was observed between the process parameters and matrix composition and it directly influenced the drug transport mechanism. In conclusion, the hydrolyzed polyacrylamide grafted carboxymethylxyloglucan holds an immense potential to be explored pharmaceutically as new matrix material for the design of targeted drug delivery system. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. EVALUATION OF SUGARCANE BAGASSE ACID HYDROLYZATE TREATMENTS FOR XYLITOL PRODUCTION

    Directory of Open Access Journals (Sweden)

    P.V. GURGEL

    1998-09-01

    Full Text Available Acid sugarcane bagasse hydrolyzate was submitted to pH shifts in order to remove toxic compounds from the medium. The hydrolyzate was treated with bases containing mono-, di- or tri-valent cations and H2SO4, and its performance as a fermentation medium was evaluated by the production of xylitol by Candida guilliermondii FTI 20037. The use of bases containing mono-valent cations was not an efficient method of detoxification, and the use of a tri-valent cation did not show any detectable improvement in detoxification. The treated hydrolyzate recovery (in volume is greatly affected by the utilized base. Treatment using Al(OH3 and NaOH showed the best hydrolyzate recovery (87.5%, while the others presented a recovery of about 45% of the original hydrolyzate volume. Considering the whole process, best results were achieved by treatment using Al(OH3 and NaOH which allowed 0.55 g of xylitol produced from each gram of xylose in the raw hydrolyzate.

  2. [Contact urticaria induced by hydrolyzed wheat proteins in cosmetics].

    Science.gov (United States)

    Olaiwan, A; Pecquet, C; Mathelier-Fusade, P; Francès, C

    2010-04-01

    Hydrolyzed wheat protein, produced by hydrolysis of gluten, is used in certain cosmetics and foods as emulsifiers and stabilizers. It can induce contact urticaria to cosmetics and/or anaphylaxis to food via an immunologic mechanism. A 28-year-old female beautician presented recurrent contact urticaria, initially on the hands and then more diffused, immediately after applying cosmetics of the same brand containing hydrolyzed wheat protein. Skin tests were positive with the cosmetics and with the hydrolyzed wheat protein contained therein. A 34-year-old woman presented four episodes of generalized urticaria after eating industrially prepared foods. She had also experienced contact urticaria with cosmetics. Skin tests with hydrolyzed wheat protein were positive. For both patients, withdrawal of cosmetics and foods containing hydrolyzed wheat protein led to the regression of symptoms. They were both tolerant to traditional wheat products, such as bread and pastries. Although contact urticaria to hydrolyzed wheat protein is rarely described, it must be understood since treatment by eradication of this product is simple and because contact urticaria may precede food allergy. Patients are tolerant to products containing unmodified wheat protein. 2010 Elsevier Masson SAS. All rights reserved.

  3. Ionotropic ATP receptors in neuronal-glial communication.

    Science.gov (United States)

    Lalo, Ulyana; Verkhratsky, Alexei; Pankratov, Yuri

    2011-04-01

    In the central nervous system ATP is released from both neurones and astroglial cells acting as a homo- and heterocellular neurotransmitter. Glial cells express numerous purinoceptors of both ionotropic (P2X) and metabotropic (P2Y) varieties. Astroglial P2X receptors can be activated by ongoing synaptic transmission and can mediate fast local signalling through elevation in cytoplasmic Ca(2+) and Na(+) concentrations. These ionic signals can be translated into various physiological messages by numerous pathways, including release of gliotransmitters, metabolic support of neurones and regulation of activity of postsynaptic glutamate and GABA receptors. Ionotropic purinoceptors represent a novel pathway of glia-driven modulation of synaptic signalling that involves the release of ATP from neurones and astrocytes followed by activation of P2X receptors which can regulate synaptic activity by variety of mechanisms expressed in both neuronal and glial compartments. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes.

    Science.gov (United States)

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

    2011-06-08

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

  5. Fine-tuned ATP signals are acute mediators in osteocyte mechanotransduction.

    Science.gov (United States)

    Kringelbach, Tina M; Aslan, Derya; Novak, Ivana; Ellegaard, Maria; Syberg, Susanne; Andersen, Christina K B; Kristiansen, Kim A; Vang, Ole; Schwarz, Peter; Jørgensen, Niklas R

    2015-12-01

    Osteocytes are considered the primary mechanosensors of bone, but the signaling pathways they apply in mechanotransduction are still incompletely investigated and characterized. A growing body of data strongly indicates that P2 receptor signaling among osteoblasts and osteoclasts has regulatory effects on bone remodeling. Therefore, we hypothesized that ATP signaling is also applied by osteocytes in mechanotransduction. We applied a short fluid pulse on MLO-Y4 osteocyte-like cells during real-time detection of ATP and demonstrated that mechanical stimulation activates the acute release of ATP and that these acute ATP signals are fine-tuned according to the magnitude of loading. ATP release was then challenged by pharmacological inhibitors, which indicated a vesicular release pathway for acute ATP signals. Finally, we showed that osteocytes express functional P2X2 and P2X7 receptors and respond to even low concentrations of nucleotides by increasing intracellular calcium concentration. These results indicate that in osteocytes, vesicular ATP release is an acute mediator of mechanical signals and the magnitude of loading. These and previous results, therefore, implicate purinergic signaling as an early signaling pathway in osteocyte mechanotransduction. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. ATP as a biological hydrotrope.

    Science.gov (United States)

    Patel, Avinash; Malinovska, Liliana; Saha, Shambaditya; Wang, Jie; Alberti, Simon; Krishnan, Yamuna; Hyman, Anthony A

    2017-05-19

    Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells. Copyright © 2017, American Association for the Advancement of Science.

  7. Hydrolyzed infant formula and early β-cell autoimmunity

    DEFF Research Database (Denmark)

    Knip, Mikael; Åkerblom, Hans K; Becker, Dorothy

    2014-01-01

    IMPORTANCE: The disease process leading to clinical type 1 diabetes often starts during the first years of life. Early exposure to complex dietary proteins may increase the risk of β-cell autoimmunity in children at genetic risk for type 1 diabetes. Extensively hydrolyzed formulas do not contain...... intact proteins. OBJECTIVE: To test the hypothesis that weaning to an extensively hydrolyzed formula decreases the cumulative incidence of diabetes-associated autoantibodies in young children. DESIGN, SETTING, AND PARTICIPANTS: A double-blind randomized clinical trial of 2159 infants with HLA......-conferred disease susceptibility and a first-degree relative with type 1 diabetes recruited from May 2002 to January 2007 in 78 study centers in 15 countries; 1078 were randomized to be weaned to the extensively hydrolyzed casein formula and 1081 were randomized to be weaned to a conventional cows' milk...

  8. 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.

  9. 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

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

    Science.gov (United States)

    Talwalkar, P G; Sreenivas, C G; Gulati, Ashish; Baxi, Hemang

    2013-07-01

    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 strong emphasis would

  11. Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP.

    Science.gov (United States)

    Hogarth, D Kyle; Sandbo, Nathan; Taurin, Sebastien; Kolenko, Vladimir; Miano, Joseph M; Dulin, Nickolai O

    2004-08-01

    Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM-alpha-actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity.

  12. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants.

    Science.gov (United States)

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    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 roles 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 Ca(2+) 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 future research on extracellular ATP as a DAMP signal.

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  14. ATP synthase from slow and fast growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

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

    2010-01-01

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

  15. Investigation of viscosity of whole hydrolyze sweetened condensed milk

    Directory of Open Access Journals (Sweden)

    O. Kalinina

    2015-05-01

    Full Text Available Introduction. Рaper is aimed at developing of low-lactose (hydrolyzed sweetened condensed milk products technology for lactose intolerant people and for the whole population. Materials and methods: Rheological characteristics were determined on a Reotest device by the 2 nd method of viscometry Results and discussion. Reasonability of ß-galactosidase use for milk lactose hydrolyze during the production of canned products with sugar was proved in the previous works. This technology gives possibility to increase the quality of condensed canned foods, to reduce sugar concentration till 50 %, to increase dietary properties. Due to the reducing of saccharose mass part till 22 and 31 % the products had a liquid consistency that’s why was a necessity to increase the viscosity properties of condensed products. One of method to increase the product viscosity is inoculation of stabilization systems. Reasonability of the usage of stabilization system Bivicioc 1L was proved. The researches of viscosity determination in whole hydrolyzed sweetened condensed milk were shown in the work. Relations of viscosity of whole hydrolyzed condensed milk to the deformation rate were presented. Conclusions Viscosity indices of experimental samples in the fresh produced products and during storage are determined and justified.

  16. 40 CFR 721.4585 - Lecithins, phospholipase A2-hydrolyzed.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Lecithins, phospholipase A2-hydrolyzed. 721.4585 Section 721.4585 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4585 Lecithins,...

  17. 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.

  18. Production of Polyhydroxyalkanoates Using Hydrolyzates of Spruce Sawdust: Comparison of Hydrolyzates Detoxification by Application of Overliming, Active Carbon, and Lignite

    Science.gov (United States)

    Kucera, Dan; Benesova, Pavla; Ladicky, Peter; Pekar, Miloslav; Sedlacek, Petr; Obruca, Stanislav

    2017-01-01

    Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the price of the carbon source used in the fermentation. Burkholderia cepacia and Burkholderia sacchari are generally considered promising candidates for PHA production from lignocellulosic hydrolyzates. The wood waste biomass has been subjected to hydrolysis. The resulting hydrolyzate contained a sufficient amount of fermentable sugars. Growth experiments indicated a strong inhibition by the wood hydrolyzate. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification. All methods of detoxification had a positive influence on the growth of biomass and PHB production. Furthermore, lignite was identified as a promising alternative sorbent which can be used for detoxification of lignocellulose hydrolyzates. Detoxification using lignite instead of activated carbon had lower inhibitor removal efficiency, but greater positive impact on growth of the bacterial culture and overall PHA productivity. Moreover, lignite is a significantly less expensive adsorbent in comparison with activated charcoal and; moreover, used lignite can be simply utilized as a fuel to, at least partially, cover heat and energetic demands of fermentation, which should improve the economic feasibility of the process. PMID:28952532

  19. Production of Polyhydroxyalkanoates Using Hydrolyzates of Spruce Sawdust: Comparison of Hydrolyzates Detoxification by Application of Overliming, Active Carbon, and Lignite

    Directory of Open Access Journals (Sweden)

    Dan Kucera

    2017-05-01

    Full Text Available Polyhydroxyalkanoates (PHAs are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the price of the carbon source used in the fermentation. Burkholderia cepacia and Burkholderia sacchari are generally considered promising candidates for PHA production from lignocellulosic hydrolyzates. The wood waste biomass has been subjected to hydrolysis. The resulting hydrolyzate contained a sufficient amount of fermentable sugars. Growth experiments indicated a strong inhibition by the wood hydrolyzate. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification. All methods of detoxification had a positive influence on the growth of biomass and PHB production. Furthermore, lignite was identified as a promising alternative sorbent which can be used for detoxification of lignocellulose hydrolyzates. Detoxification using lignite instead of activated carbon had lower inhibitor removal efficiency, but greater positive impact on growth of the bacterial culture and overall PHA productivity. Moreover, lignite is a significantly less expensive adsorbent in comparison with activated charcoal and; moreover, used lignite can be simply utilized as a fuel to, at least partially, cover heat and energetic demands of fermentation, which should improve the economic feasibility of the process.

  20. Production of Polyhydroxyalkanoates Using Hydrolyzates of Spruce Sawdust: Comparison of Hydrolyzates Detoxification by Application of Overliming, Active Carbon, and Lignite.

    Science.gov (United States)

    Kucera, Dan; Benesova, Pavla; Ladicky, Peter; Pekar, Miloslav; Sedlacek, Petr; Obruca, Stanislav

    2017-05-28

    Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the price of the carbon source used in the fermentation. Burkholderia cepacia and Burkholderia sacchari are generally considered promising candidates for PHA production from lignocellulosic hydrolyzates. The wood waste biomass has been subjected to hydrolysis. The resulting hydrolyzate contained a sufficient amount of fermentable sugars. Growth experiments indicated a strong inhibition by the wood hydrolyzate. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification. All methods of detoxification had a positive influence on the growth of biomass and PHB production. Furthermore, lignite was identified as a promising alternative sorbent which can be used for detoxification of lignocellulose hydrolyzates. Detoxification using lignite instead of activated carbon had lower inhibitor removal efficiency, but greater positive impact on growth of the bacterial culture and overall PHA productivity. Moreover, lignite is a significantly less expensive adsorbent in comparison with activated charcoal and; moreover, used lignite can be simply utilized as a fuel to, at least partially, cover heat and energetic demands of fermentation, which should improve the economic feasibility of the process.

  1. 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

  2. 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.

  3. Mitochondrial function is required for extracellular ATP-induced NLRP3 inflammasome activation.

    Science.gov (United States)

    Sadatomi, Daichi; Nakashioya, Kazutaka; Mamiya, Sayaka; Honda, Shino; Kameyama, Yuka; Yamamura, Yasuo; Tanimura, Susumu; Takeda, Kohsuke

    2017-06-01

    The NLRP3 inflammasome plays a critical role in the processing and release of inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18. Accumulating evidence suggests that mitochondria are common mediators of NLRP3 inflammasome activation induced by a wide range of inflammatory stimuli; however, the precise role of mitochondria is still not fully understood. Here, we show that mitochondrial function is required for extracellular ATP-induced NLRP3 inflammasome activation. Extracellular ATP induced the loss of mitochondrial membrane potential and mitochondrial fragmentation in a different manner than other stimuli in primary mouse macrophages. CCCP, an uncoupler and antimycin A, an inhibitor of the mitochondrial electron transport chain, inhibited IL-1β release induced by ATP but not by other stimuli. CCCP did not inhibit the ATP-induced generation of reactive oxygen species and cell death, both of which are known to promote IL-1β release, but did inhibit the ATP-induced activation of caspase-1, a component of the NLRP3 inflammasome. These results suggest that mitochondrial function is required somewhat specifically for ATP-induced NLRP3 inflammasome activation. In contrast to many previous reports that dysfunctional mitochondria promote NLRP3 inflammasome activation, the function of intact mitochondria appears to be required for NLRP3 inflammasome activation, depending on the stimulus. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

    Science.gov (United States)

    Alexandre, Adolfo; Reynafarje, Baltazar; Lehninger, Albert L.

    1978-01-01

    In order to verify more directly our earlier measurements showing that, on the average, close to four vectorial H+ are rejected per pair of electrons passing each of the three energy-conserving sites of the mitochondrial electron transport chain, direct tests of the H+/2e- ratio for sites 2 and 3 were carried out in the presence of permeant charge-compensating cations. Site 2 was examined by utilizing succinate as electron donor and ferricyanide as electron acceptor from mitochondrial cytochrome c; the directly measured H+/2e- ratio was close to 4. Energy-conserving site 3 was isolated for study with ferrocyanide or ascorbate plus tetramethylphenylenediamine as electron donors to cytochrome c and with oxygen as electron acceptor. The directly measured H+/2e- ratio for site 3 was close to 4. The H+/ATP ratio (number of vectorial H+ ejected per ATP hydrolyzed) was determined with a new method in which the steady-state rates of both H+ ejection and ATP hydrolysis were measured in the presence of K+ + valinomycin. The H+/ATP ratio was found to approach 3.0. A proton cycle for oxidative phosphorylation is proposed, in which four electrochemical H+ equivalents are ejected per pair of electrons passing each energy-conserving site; three of the H+ equivalents pass inward to derive ATP synthesis from ADP and phosphate and the fourth H+ is used to bring about the energy-requiring electrogenic expulsion of ATP4- in exchange for extramitochondrial ADP3-, via the H+/H2PO4- symporter. PMID:31621

  5. Surface tension and stability of foams based of keratin hydrolyzate

    Directory of Open Access Journals (Sweden)

    Zhanar Ospanova

    2015-03-01

    Full Text Available The protein obtained in the alkali hydrolysis consist of amino acid residues that are natural macromolecular surfactants and they can may be used as effective foam stabilizers. The features of dynamic surface tension and stability of foams on based of aqueous solutions of keratin hydrolyzate in a concentration range of 1-10% were studied. The relaxation time – of the adsorption layers of keratin hydrolyzate is equaled to 10-12 min. The parameters of adsorption at the liquid – gas interface were defined. The maximum surface activity, foaming and foam stability corresponds to a neutral pH close to isoelectric state of the protein. Increase the foam stability at pH ~ 7 proceeds due to the conformational changes of macro-molecules of the protein at the interface liquid – gas, forming particles of colloidal size, clogging channels Plateau-Gibbs and preventing expiration of the liquid film between.

  6. Green Tea Polyphenols Decrease Strecker Aldehydes and Bind to Proteins in Lactose-Hydrolyzed UHT Milk.

    Science.gov (United States)

    Jansson, Therese; Rauh, Valentin; Danielsen, Bente P; Poojary, Mahesha M; Waehrens, Sandra S; Bredie, Wender L P; Sørensen, John; Petersen, Mikael A; Ray, Colin A; Lund, Marianne N

    2017-12-06

    The effect of epigallocatechin gallate enriched green tea extract (GTE) on flavor, Maillard reactions and protein modifications in lactose-hydrolyzed (LH) ultrahigh temperature (UHT) processed milk was examined during storage at 40 °C for up to 42 days. Addition of GTE inhibited the formation of Strecker aldehydes by up to 95% compared to control milk, and the effect was similar when GTE was added either before or after UHT treatment. Release of free amino acids, caused by proteolysis, during storage was also decreased in GTE-added milk either before or after UHT treatment compared to control milk. Binding of polyphenols to milk proteins was observed in both fresh and stored milk samples. The inhibition of Strecker aldehyde formation by GTE may be explained by two different mechanisms; inhibition of proteolysis during storage by GTE or binding of amino acids and proteins to the GTE polyphenols.

  7. Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals

    DEFF Research Database (Denmark)

    Andreasen, Mette Findal; Kroon, P A; Williamson, G

    2001-01-01

    Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets...... and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary...... from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye...

  8. Rheological Properties of Partially Hydrolyzed Polyacrylamide Seeded by Nanoparticles

    OpenAIRE

    Hu, Z; Haruna, M; Gao, H; Nourafkan, E; Wen, D

    2017-01-01

    This work aims to improve the rheological properties of partially hydrolyzed polyacrylamide (HPAM) for enhanced oil recovery by using silica (or silicon dioxide, SiO₂) nanoparticles (NPs). Novel aqueous HPAM-based SiO₂ nanocomposites were formulated, and their rheological properties were investigated under different salinities, temperatures, and aging times. The results show that the inclusion of silica NPs significantly improved the viscosity and viscoelastic properties of HPAM especially un...

  9. Dipeptidyl peptidase IV inhibitory activity of protein hydrolyzates ...

    African Journals Online (AJOL)

    Dipeptidyl peptidase IV inhibitory activity of protein hydrolyzates from Amaranthus hypochondriacus L. Grain and their influence on postprandial glycemia in Streptozotocin-induced diabetic mice. S-S Jorge, R-B Raúl, G-L Isabel, P-A Edith, E-BH Bernardo, A-PJ César, D-G Gerardo, R-R Rubén ...

  10. Hydrolyzed collagen interferes with in vitro photoprotective effectiveness of sunscreens

    Directory of Open Access Journals (Sweden)

    Daniela D'Almeida Peres

    2017-06-01

    Full Text Available ABSTRACT The chronological skin aging is a progressive and natural process with genetic and physiological changes. However, ultraviolet (UV radiation may accelerate the oxidative stress, generating carcinogenesis and photoaging. Natural compounds and their applications are considered a trend in the cosmetic market. The protein-based film-forming compounds play an important role, once it collaborates for the better distribution of sunscreens on the skin. Here we investigated the in vitro photoprotective effectiveness of sunscreens containing the hydrolyzed collagen associated with UVA, UVB and/or inorganic filters. Sunscreens were developed with octocrylene (7.5%, butyl methoxydibenzoylmethane (avobenzone (3.0% and/or titanium dioxide (5.0%, associated or not with the hydrolyzed collagen (3.0%. In vitro photoprotective effectiveness was determined in a Labsphere(r UV2000S by the establishment of the sun protection factor (SPF and critical wavelength (nm values. Physicochemical and organoleptic characteristics were also assayed. The hydrolyzed collagen subjectively improved the formulation sensory characteristics. However, this bioactive compound led to a decrease of the SPF values of the photoprotective formulations containing octocrylene alone and octocrylene + butyl methoxydibenzoylmethane + TiO2. This inadequate interaction may be considered during the development of new sunscreens intended to contain protein-based components.

  11. Students' Interdisciplinary Reasoning about "High-Energy Bonds" and ATP

    CERN Document Server

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  13. Extracellular ATP is a key modulator of alveolar bone loss in periodontitis.

    Science.gov (United States)

    Binderman, Itzhak; Gadban, Nasir; Yaffe, Avinoam

    2017-09-01

    Periodontal diseases are initiated by pathogenic bacterial biofilm activity that induces a host inflammatory cells immune response, degradation of dento gingival fibrous tissue and its detachment from root cementum. It is well accepted, that osteoclastic alveolar bone loss is governed exclusively through secretion of proinflammatory cytokines. Nevertheless, our findings suggest that once degradation of collagen fibers by MMPs occurs, a drop of cellular strains cause immediate release of ATP from marginal gingival fibroblasts, cell deformation and influx of Ca+2. Increased extracellular ATP (eATP) by interacting with P2×7 purinoreceptors, present on fibroblasts and osteoblasts, induces generation of receptor activator of nuclear factor kB ligand (RANKL) that further activates osteoclastic alveolar bone resorption and bone loss. In addition, increased eATP levels may amplify inflammation by promoting leukocyte recruitment and NALP3-inflammasome activation via P2×7. Then, the inflammatory cells secrete cytokines, interleukin IL-1, TNF and RANKL that further trigger alveolar bone resorption. Moreover, eATP can be secreted from periodontal bacteria that may further contribute to inflammation and bone loss in periodontitis. It seems therefore, that eATP is a key modulator that initiates the pathway of alveolar bone resorption and bone loss in patients with periodontal disease. In conclusion, we propose that strain release in gingival fibroblasts aligned on collagen fibers, due to activity of MMP, activates release of ATP that triggers the pathway of alveolar bone resorption in periodontitis. We predict that by controlling the eATP interaction with its cellular purinoreceptors will reduce significantly bone loss in periodontitis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells.

    Science.gov (United States)

    Akopova, Irina; Tatur, Sabina; Grygorczyk, Mariusz; Luchowski, Rafał; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian; Grygorczyk, Ryszard

    2012-03-01

    Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

  15. Glucose and the ATP paradox in yeast.

    NARCIS (Netherlands)

    Somsen, O.J.G.; Hoeben, M.A.; Esgalhado, M.E.L.M.; Snoep, J.L.; Visser, D.; van der Heijden, R.T.J.M.; Heijnen, J.J.; Westerhoff, H.V.

    2000-01-01

    A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be

  16. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    aliquots of standards increase quality control and ease daily operation. The medium (Lumin(PM) buffer, tap water or MilliQ water) for preparation of ATP-standard dilution significantly affected the rlu response of the ATP-standard dilutions (20% difference). The effect of dilution media and of sample...

  17. Asymmetric ATP Binding and Hydrolysis Activity of the Thermus aquaticus MutS Dimer Is Key to Modulation of Its Interactions with Mismatched DNA†

    Science.gov (United States)

    Antony, Edwin; Hingorani, Manju M.

    2010-01-01

    Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2–Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30–50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1ADP–S2ATP is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1ATP–S2ATP and S1ATP–S2ADP are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair. PMID:15476405

  18. Adenosine 5′-triphosphate (ATP supplements are not orally bioavailable: a randomized, placebo-controlled cross-over trial in healthy humans

    Directory of Open Access Journals (Sweden)

    Arts Ilja CW

    2012-04-01

    Full Text Available Abstract Background Nutritional supplements designed to increase adenosine 5′-triphosphate (ATP concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity exercise. Oral ATP supplements have beneficial effects in some but not all studies examining physical performance. One of the remaining questions is whether orally administered ATP is bioavailable. We investigated whether acute supplementation with oral ATP administered as enteric-coated pellets led to increased concentrations of ATP or its metabolites in the circulation. Methods Eight healthy volunteers participated in a cross-over study. Participants were given in random order single doses of 5000 mg ATP or placebo. To prevent degradation of ATP in the acidic environment of the stomach, the supplement was administered via two types of pH-sensitive, enteric-coated pellets (targeted at release in the proximal or distal small intestine, or via a naso-duodenal tube. Blood ATP and metabolite concentrations were monitored by HPLC for 4.5 h (naso-duodenal tube or 7 h (pellets post-administration. Areas under the concentration vs. time curve were calculated and compared by paired-samples t-tests. Results ATP concentrations in blood did not increase after ATP supplementation via enteric-coated pellets or naso-duodenal tube. In contrast, concentrations of the final catabolic product of ATP, uric acid, were significantly increased compared to placebo by ~50% after administration via proximal-release pellets (P = 0.003 and naso-duodenal tube (P = 0.001, but not after administration via distal-release pellets. Conclusions A single dose of orally administered ATP is not bioavailable, and this may explain why several studies did not find ergogenic effects of oral ATP supplementation. On the other hand, increases in uric acid after release of

  19. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured...... the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency...... for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A...

  20. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation...

  1. 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...

  2. Microstructural study of pre-treated and enzymatic hydrolyzed bamboo

    Directory of Open Access Journals (Sweden)

    Funsho O. KOLAWOLE

    2016-07-01

    Full Text Available Bamboo was used as biomass feedstock which was pre-treated using dilute acid hydrolysis followed by enzymatic hydrolysis. The bamboo was mechanical ground to particle sizes 212–500µm, followed by pre-treatment with dilute sulfuric acid at a concentration of 0.5 and 1.0 (%v/v at temperatures of 25, 110, 120, 150 and 200°C with time intervals of 2 and 4 hours. Pre-hydrolyzate was later analyzed for reducing sugar using UV-Vis spectrophotometry. Under the above conditions, a maximum glucose yield of 153.1 mg/g was obtained at 200°C and acid concentrations of 1% for 4 hours. Water insoluble solids obtained were subsequently hydrolyzed with Celluclast (Trichoderma reesi and β-glucosidase (Novozyme 188 for 72 hours. Optical Microscope and ESEM images of bamboo samples were obtained at various stages of pre-treatment and enzymatic hydrolysis. Result reveals a breakdown in the ligno-cellulosic structure of the bamboo during exposure to dilute acid and enzymatic hydrolysis.

  3. Yeast Biomass Production in Brewery's Spent Grains Hemicellulosic Hydrolyzate

    Science.gov (United States)

    Duarte, Luís C.; Carvalheiro, Florbela; Lopes, Sónia; Neves, Ines; Gírio, Francisco M.

    Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery's spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h-1, 0.61 g g-1, and 0.56 g 1-1 h-1, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.

  4. ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

    Science.gov (United States)

    Kourie, J I

    1997-05-01

    Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C) coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl- channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive Cl- channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current amplitude, Imax and integral current, I') and kinetic parameters (frequency of opening FO, probability of the channel being open PO, mean open TO and closed Tc times) show a nonspecific block of the voltage- and Ca2+-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel. The SCl channel block was not due to Ca2+ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg2+. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP4- > ADP3- > > > AMP2-. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel by [ATP]cis and [Ca2+]cis indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent cycling of the Ca2+ between the SR and the sarcoplasm.

  5. Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling.

    Science.gov (United States)

    Moehring, Francie; Cowie, Ashley M; Menzel, Anthony D; Weyer, Andy D; Grzybowski, Michael; Arzua, Thiago; Geurts, Aron M; Palygin, Oleg; Stucky, Cheryl L

    2018-01-16

    The first point of our body's contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions. © 2018, Moehring et al.

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

    Directory of Open Access Journals (Sweden)

    Lina Wang

    2015-01-01

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

  7. Modulation of extracellular ATP content of mast cells and DRG neurons by irradiation: studies on underlying mechanism of low-level-laser therapy.

    Science.gov (United States)

    Wang, Lina; Hu, Lei; Grygorczyk, Ryszard; Shen, Xueyong; Schwarz, Wolfgang

    2015-01-01

    Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca(2+)]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

  8. Extracellular ATP Induces Calcium Signaling in Odontoblasts.

    Science.gov (United States)

    Lee, B M; Jo, H; Park, G; Kim, Y H; Park, C K; Jung, S J; Chung, G; Oh, S B

    2017-02-01

    Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca(2+) concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca(2+)-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X

  9. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Lenartowicz Malgorzata

    2016-08-01

    Full Text Available Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD or the rare autosomal disorder Wilson disease (WD, respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation.

  10. The Effects of Wheat Bran Composition on the Production of Biomass-Hydrolyzing Enzymes by Penicillium decumbens

    Science.gov (United States)

    Sun, Xianyun; Liu, Ziyong; Qu, Yinbo; Li, Xuezhi

    The effects of the starch, protein, and soluble oligosaccharides contents in wheat bran on the extracellular biomass-hydrolyzing enzymes activities released by Penicillium decumbens mycelia grown in batch fermentations have been examined. The results showed increased starch content correlated directly with an increase in released amylase activity but inversely with the levels of secreted cellulase and xylanase. High amounts of protein in wheat bran also reduced the activities of cellulase, xylanase and protease in the culture medium. The effects of the soluble and insoluble components of wheat bran and cello-oligosaccharides supplements on production of extracellular cellulase and xylanase were compared. The soluble cello-oligosaccharides compositions in wheat bran were proved to be one of the most significant factors for cellulase production. According to the results of this research, determining and regulating the composition of wheat bran used as a fermentation supplement may allow for improved induction of cellulase and xylanase production.

  11. Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia.

    Science.gov (United States)

    Alberici, Luciane C; Paim, Bruno A; Zecchin, Karina G; Mirandola, Sandra R; Pestana, Cezar R; Castilho, Roger F; Vercesi, Anibal E; Oliveira, Helena C F

    2013-06-14

    We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoK(ATP) protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoK(ATP) activity and evaluate the influence of this trait on cell redox state and viability. Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. HTG mice mononuclear cells displayed increased mitoK(ATP) activity, as evidenced by higher resting respiration rates that were sensitive to mitoK(ATP) antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoK(ATP) further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoK(ATP) channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoK(ATP) channels. Thus, mitoK(ATP) opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

  12. Initial rate and equilibrium isotope exchange studies on the ATP-dependent activity of polyphosphate Glucokinase from Propionibacterium shermanii.

    Science.gov (United States)

    Kowalczyk, T H; Horn, P J; Pan, W H; Phillips, N F

    1996-05-28

    Polyphosphate glucokinase [EC 2.7.1.63] catalyzes the phosphorylation of glucose using either inorganic polyphosphate [poly(P)] or ATP as the phosphoryl donor. Both activities purified from Propionibacterium shermanii are the functional properties of a single enzyme with separate binding sites for the two phosphoryl donor substrates. The enzyme was found to utilize poly(P) much more efficiently than it does ATP, with a kcat/Kpoly(P) to kcat/KATP ratio of 2800. The catalytic constant for poly(P) is about 2-fold higher than for ATP. Other nucleotides like GTP and dATP also served as substrates with good efficiencies. The ATP-dependent reaction was analyzed using steady-state kinetics and isotopic exchange kinetics at chemical equilibrium. Intersecting initial velocity patterns for both glucose and ATP indicate sequential addition of substrates. Product inhibition studies resulted in two competitive and two noncompetitive patterns, which is characteristic of a Theorell-Chance mechanism or a random mechanism with two dead-end complexes. Results of isotope exchange experiments, however, rule out a Theorell-Chance mechanism, as well as a truly random mechanism. They are not consistent with a partially random mechanism (although a kinetically compulsory order of substrate binding is not excluded), where glucose is preferentially bound to free enzyme before ATP, and ADP is preferentially released as the first product, followed by glucose 6-phosphate. Dead-end inhibition analysis confirms this order of substrate binding. Competitive inhibition of ADP vs ATP is explained as resulting primarily from binding as a dead-end inhibitor (E.Glc.ADP) and not as a product. Another weaker abortive complex, E.ATP.G6P, is also formed. The chemical transformation or the release of ADP is the rate-limiting step in ATP utilization.

  13. 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

  14. CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

    Science.gov (United States)

    Long, Jingdong; Lei, Xiaolu; Chen, Meiyun; Yang, Shulei; Sun, Tao; Zeng, Junwei; Yu, Deqian; Tian, Hong; Liu, Xiaohong

    2017-11-10

    Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening

  15. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  16. A new myofilament contraction model with ATP consumption for ventricular cell model.

    Science.gov (United States)

    Muangkram, Yuttamol; Noma, Akinori; Amano, Akira

    2017-08-02

    A new contraction model of cardiac muscle was developed by combining previously described biochemical and biophysical models. The biochemical component of the new contraction model represents events in the presence of Ca(2+)-crossbridge attachment and power stroke following inorganic phosphate release, detachment evoked by the replacement of ADP by ATP, ATP hydrolysis, and recovery stroke. The biophysical component focuses on Ca(2+) activation and force (F b) development assuming an equivalent crossbridge. The new model faithfully incorporates the major characteristics of the biochemical and biophysical models, such as F b activation by transient Ca(2+) ([Ca(2+)]-F b), [Ca(2+)]-ATP hydrolysis relations, sarcomere length-F b, and F b recovery after jumps in length under the isometric mode and upon sarcomere shortening after a rapid release of mechanical load under the isotonic mode together with the load-velocity relationship. ATP consumption was obtained for all responses. When incorporated in a ventricular cell model, the contraction model was found to share approximately 60% of the total ATP usage in the cell model.

  17. Technological aspects of lactose-hydrolyzed milk powder.

    Science.gov (United States)

    Torres, Jansen Kelis Ferreira; Stephani, Rodrigo; Tavares, Guilherme M; de Carvalho, Antônio Fernandes; Costa, Renata Golin Bueno; de Almeida, Carlos Eduardo Rocha; Almeida, Mariana Ramos; de Oliveira, Luiz Fernando Cappa; Schuck, Pierre; Perrone, Ítalo Tuler

    2017-11-01

    Few reports describe the effect of lactose hydrolysis on the properties of milk powder during production and storage. Hence, the aim of this study was to evaluate the effects of five different levels of enzymatic lactose hydrolysis during the production and storage of milk powder. As the lactose hydrolysis rate increased, adhesion to the drying chamber also increased, due to higher levels of particle agglomeration. Additionally, more brown powder was obtained when the lactose hydrolysis rate was increased, which in turn negatively affected rehydration ability. Using Raman spectroscopy, crystallization of the lactose residues in various samples was assessed over 6weeks of accelerated aging at a room temperature environment with 75.5% of air moisture. Products with 25% or greater lactose hydrolysis showed no signs of crystallization, in contrast to the non-hydrolyzed sample. Copyright © 2017. Published by Elsevier Ltd.

  18. Hydrolyzed polyacrylamide biodegradation and mechanism in sequencing batch biofilm reactor.

    Science.gov (United States)

    Yan, Miao; Zhao, Lanmei; Bao, Mutai; Lu, Jinren

    2016-05-01

    An investigation was performed to study the performance of a sequencing batch biofilm reactor (SBBR) to treat hydrolyzed polyacrylamides (HPAMs) and to determine the mechanisms of HPAM biodegradation. The mechanisms for the optimized parameters that significantly improved the degradation efficiency of the HPAMs were investigated by a synergistic effect of the co-metabolism in the sludge and the enzyme activities. The HPAM and TOC removal ratio reached 54.69% and 70.14%. A significant decrease in the total nitrogen concentration was measured. The carbon backbone of the HPAMs could be degraded after the separation of the amide group according to the data analysis. The HPLC results indicated that the HPAMs could be converted to polymer fragments without the generation of the acrylamide monomer intermediate. The results from high-throughput sequencing analysis revealed proteobacterias, bacteroidetes and planctomycetes were the key microorganisms involved in the degradation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Thermal characterization of partially hydrolyzed cassava (Manihot esculenta starch granules

    Directory of Open Access Journals (Sweden)

    Luiz Gustavo Lacerda

    2008-12-01

    Full Text Available Cassava starch, partially hydrolyzed by fungal á-amylase, was characterized using thermal analysis, light microscopy and X-ray diffraction. Thermal degradation was initiated at lower degradation temperatures after enzymatic treatment and the DSC (Differential scanning calorimetry analysis showed almost similar range of gelatinization temperature, but the enthalpies of gelatinization were quite increased for the partially hydrolyzed starch granules. The results suggested that the partial degradation of the starch granules was concentrated in the amorphous regions.Amilases fúngicas são comumente empregadas a amidos com o intuito de otimizar o rendimento de leveduras, modificar a textura de produtos panificados e prolongar a vida de prateleira do produto final. A hidrólise parcial enzimática pode auxiliar no entendimento da estrutura do amido ganular. Amido de mandioca parcialmente hidrolisado por á-amilase fúngica foi investigado utilizando-se técnicas termoanalíticas, microscopia ótica e difratometria por raios X. A degradação térmica iniciou-se a temperaturas menores após o tratamento enzimático e a análise por DSC mostrou uma próxima faixa de temperatura de gelatinização, porém, a entalpia necessária para o evento foi maior para os grânulos parcialmente hidrolisados. Os resultados sugerem que a degradação parcial do amido granular foi concentrada em regiões amorfas.

  20. PYK2 mediates BzATP-induced extracellular matrix proteins synthesis.

    Science.gov (United States)

    Torigoe, Go; Nagao, Mayu; Tanabe, Natsuko; Manaka, Soichiro; Kariya, Taro; Kawato, Takayuki; Sekino, Jumpei; Kato, Shunichiro; Maeno, Masao; Suzuki, Naoto; Shimizu, Noriyoshi

    2017-12-16

    Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-01

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

  2. 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.

  3. 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. PMID:22485177

  4. Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

    Directory of Open Access Journals (Sweden)

    Leigh A. Swayne

    2017-08-01

    Full Text Available Pannexin 1 (Panx1 channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs. Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

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

    Science.gov (United States)

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

    2014-09-01

    ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Effect of endosperm hardness on an ethanol process using a granular starch hydrolyzing enzyme

    Science.gov (United States)

    Granular starch hydrolyzing enzymes (GSHE) can hydrolyze starch at low temperature (32 deg C). The dry grind process using GSHE (GSH process) has fewer unit operations and no changes in process conditions (pH 4.0 and 32 deg C) compared to the conventional process because it dispenses with the cookin...

  7. Extracellular ATP regulates cell death of lymphocytes and monocytes induced by membrane-bound lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium.

    Science.gov (United States)

    Into, Takeshi; Okada, Kazutaka; Inoue, Nobuo; Yasuda, Motoaki; Shibata, Ken-ichiro

    2002-01-01

    The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT-4, and a monocytic cell line, HL-60, was upregulated by ATP added extracellularly in a dose-dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate-oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase-3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate-oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.

  8. Lipid production for biofuels from hydrolyzate of waste activated sludge by heterotrophic Chlorella protothecoides.

    Science.gov (United States)

    Wen, Qinxue; Chen, Zhiqiang; Li, Pengfei; Duan, Ran; Ren, Nanqi

    2013-09-01

    Microalga Chlorella protothecoides can accumulate high proportion of lipids during the heterotrophic growth with glucose as the carbon source. However, its commercial application is restricted due to the high cost of the carbon source. In this study, the wasted activated sludge (WAS) was hydrolyzed after ultrasonic pre-treatment and the hydrolyzate obtained was used as an alternative carbon source for algal biomass and biodiesel production. The results indicate that C. protothecoides can proliferate in the WAS hydrolyzate and accumulate biolipid. The final lipid content of the culture fed with the hydrolyzate was 21.5±1.44% (weight percent) after 156 h cultivation in flasks and the maximum biomass obtained was 0.5 g L(-1). Acetic acid and isovaleric acid were favorable carbon sources for cell growth. The soluble microbial products (SMP) presents in the hydrolyzate can also be used as a carbon source for cell growth. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

    Directory of Open Access Journals (Sweden)

    Rozan Vroman

    2014-05-01

    Full Text Available Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms, highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form

  10. Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease.

    Science.gov (United States)

    Corrêa, Ana Paula F; Daroit, Daniel J; Coelho, Julise; Meira, Stela M M; Lopes, Fernanda C; Segalin, Jéferson; Risso, Patrícia H; Brandelli, Adriano

    2011-09-01

    Bioactive peptides might be released from precursor proteins through enzymatic hydrolysis. These molecules could be potentially employed in health and food products. In this investigation, ovine milk caseinate hydrolysates obtained with a novel microbial protease derived from Bacillus sp. P7 were evaluated for antioxidant, antimicrobial, and angiotensin I-converting enzyme (ACE)-inhibitory activities. Antioxidant activity measured by the 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid method increased with hydrolysis time up to 2 h, remaining stable for up to 4 h. Hydrolysates showed low 2,2-diphenyl-1-picrylhydrazyl radical-scavenging abilities, with higher activity (31%) reached after 1 h of hydrolysis. Fe(2+) -chelating ability was maximum for 0.5 h hydrolysates (83.3%), decreasing thereafter; and the higher reducing power was observed after 1 h of hydrolysis. ACE-inhibitory activity was observed to increase up to 2 h of hydrolysis (94% of inhibition), declining afterwards. 3 h hydrolysates were shown to inhibit the growth of Bacillus cereus, Corynebacterium fimi, Aspergillus fumigatus, and Penicillium expansum. Ovine caseinate hydrolyzed with Bacillus sp. P7 protease presented antioxidant, antihypertensive, and antimicrobial activities. Hydrolysis time was observed to affect the evaluated bioactivities. Such hydrolysates might have potential applications in the food industry. Copyright © 2011 Society of Chemical Industry.

  11. Dynamic changes of substrate reactivity and enzyme adsorption on partially hydrolyzed cellulose.

    Science.gov (United States)

    Shi, Jian; Wu, Dong; Zhang, Libing; Simmons, Blake A; Singh, Seema; Yang, Bin; Wyman, Charles E

    2017-03-01

    The enzymatic hydrolysis of cellulose is a thermodynamically challenging catalytic process that is influenced by both substrate-related and enzyme-related factors. In this study, a proteolysis approach was applied to recover and clean the partially converted cellulose at the different stages of enzymatic hydrolysis to monitor the hydrolysis rate as a function of substrate reactivity/accessibility and investigate surface characteristics of the partially converted cellulose. Enzyme-substrate interactions between individual key cellulase components from wild-type Trichoderma reesei and partially converted cellulose were followed and correlated to the enzyme adsorption capacity and dynamic sugar release. Results suggest that cellobiohydrolase CBH1 (Cel7A) and endoglucanases EG2 (Cel5A) adsorption capacities decreased as cellulose was progressively hydrolyzed, likely due to the "depletion" of binding sites. Furthermore, the degree of synergism between CBH1 and EG2 varied depending on the enzyme loading and the substrates. The results provide a better understanding of the relationship between dynamic change of substrate features and the functionality of various cellulase components during enzymatic hydrolysis. Biotechnol. Bioeng. 2017;114: 503-515. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. ATP8B1 and ATP11C: Two Lipid Flippases Important for Hepatocyte Function

    NARCIS (Netherlands)

    Naik, Jyoti; de Waart, Dirk R.; Utsunomiya, Karina; Duijst, Suzanne; Mok, Kam Ho; Oude Elferink, Ronald P. J.; Bosma, Piter J.; Paulusma, Coen C.

    2015-01-01

    P4 ATPases are lipid flippases and transport phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. Lipid flipping is important for the biogenesis of transport vesicles. Recently it was shown that loss of the P4 ATPases ATP8B1 and ATP11C are associated with severe

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanism remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on limb muscle interstitial...... local concentration. Key words: sympathetic nerve activity, vasodilation, endothelium, skeletal muscle....

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

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

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

  15. The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics.

    Science.gov (United States)

    Yu, Corey H; Yang, Nan; Bothe, Jameson; Tonelli, Marco; Nokhrin, Sergiy; Dolgova, Natalia V; Braiterman, Lelita; Lutsenko, Svetlana; Dmitriev, Oleg Y

    2017-11-03

    The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. PAP and NT5E inhibit nociceptive neurotransmission by rapidly hydrolyzing nucleotides to adenosine

    Science.gov (United States)

    2011-01-01

    Background Prostatic acid phosphatase (PAP) and ecto-5'-nucleotidase (NT5E, CD73) produce extracellular adenosine from the nucleotide AMP in spinal nociceptive (pain-sensing) circuits; however, it is currently unknown if these are the main ectonucleotidases that generate adenosine or how rapidly they generate adenosine. Results We found that AMP hydrolysis, when measured histochemically, was nearly abolished in dorsal root ganglia (DRG) neurons and lamina II of spinal cord from Pap/Nt5e double knockout (dKO) mice. Likewise, the antinociceptive effects of AMP, when combined with nucleoside transport inhibitors (dipyridamole or 5-iodotubericidin), were reduced by 80-100% in dKO mice. In addition, we used fast scan cyclic voltammetry (FSCV) to measure adenosine production at subsecond resolution within lamina II. Adenosine was maximally produced within seconds from AMP in wild-type (WT) mice but production was reduced >50% in dKO mice, indicating PAP and NT5E rapidly generate adenosine in lamina II. Unexpectedly, we also detected spontaneous low frequency adenosine transients in lamina II with FSCV. Adenosine transients were of short duration (60%) in frequency in Pap-/-, Nt5e-/- and dKO mice, suggesting these ectonucleotidases rapidly hydrolyze endogenously released nucleotides to adenosine. Field potential recordings in lamina II and behavioral studies indicate that adenosine made by these enzymes acts through the adenosine A1 receptor to inhibit excitatory neurotransmission and nociception. Conclusions Collectively, our experiments indicate that PAP and NT5E are the main ectonucleotidases that generate adenosine in nociceptive circuits and indicate these enzymes transform pulsatile or sustained nucleotide release into an inhibitory adenosinergic signal. PMID:22011440

  17. Spouted bed drying efficiency of bovine hydrolyzed collagen

    Directory of Open Access Journals (Sweden)

    José Júnior Butzge

    Full Text Available Summary Bovine hydrolyzed collagen (BHC is an important food supplement normally consumed in the form of capsules or powder in order to stimulate the synthesis of collagen, promote health and assist in esthetics. The transformation of liquid foods into powders by drying is a difficult operation due to the complex physical and chemical changes resulting from the use of high temperatures, which may result in low drying efficiency and unwanted physicochemical and nutritional characteristics in the final product. In this work, a process engineering approach was used aiming to maximize the drying efficiency and investigate the potential of using a spouted bed on the drying performance of BHC. The effects of feed mode, type of inert material and use of an adjuvant on powder production efficiency were analyzed using a 23 factorial experimental design. A statistical analysis showed significant effects of all the independent variables on drying performance. The maximum powder production efficiencies were achieved using polypropylene as the inert material and atomization as the feed mode. Under the optimal process conditions, up to 85% efficiency was obtained, demonstrating that the spouted bed is a technically viable equipment for drying BHC.

  18. Functionality of whey protein isolates and hydrolyzed whey proteins

    Directory of Open Access Journals (Sweden)

    Zoran Herceg

    2005-07-01

    Full Text Available The aim of this study was to determine functional properties: solubility, dispersibility, viscosity, emulsifiying, foaming and rheological properties of hydrolyzed whey protein (HWP and whey protein isolate (WPI. Particle size analysis and specific area of HWP and WPI were performed by «Mie – theory» of «light scatering» using «Malvern Mastersizer X». The results of this analysis have shown that HWP had higher particle size and specific area than WPI.By examing functional properties, it has been established that HWP has higher solubility, dispersibility, emulsifiying properties as well as foam stability (FSI and MFS compared to WPI. Rheological properties of protein suspensions were determined in 10, 15, and 18 % suspension of proteins (HWP and WPI by rotational viscosimeter, Brookfiel DV-III at 25°C. Apparent viscosity at 200 s-1 was calculated, using Newtonian law. On the basis of measured shear rate and shear stress, using least squere method, rheological parameters, flow behavior indeks (n and consistency coefficient (k were determined according to the Ostwald de Waele model. The highest viscosity was observed in 18% HWP model system while the least viscosity was found in a model system prepared with 10% WPI.

  19. Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides.

    Science.gov (United States)

    Schmidt, Sabine; Rainieri, Sandra; Witte, Simone; Matern, Ulrich; Martens, Stefan

    2011-03-01

    Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-β-glucoside revealed considerable β-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic α- or β-glucosides identified three efficient β-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid β-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7- or 4'-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides.

  20. Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications

    Science.gov (United States)

    Khalikova, Elvira; Susi, Petri; Korpela, Timo

    2005-01-01

    Dextran is a chemically and physically complex polymer, breakdown of which is carried out by a variety of endo- and exodextranases. Enzymes in many groups can be classified as dextranases according to function: such enzymes include dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-α-glucosidases. Cycloisomalto-oligosaccharide glucanotransferase does not formally belong to the dextranases even though its side reaction produces hydrolyzed dextrans. A new classification system for glycosylhydrolases and glycosyltransferases, which is based on amino acid sequence similarities, divides the dextranases into five families. However, this classification is still incomplete since sequence information is missing for many of the enzymes that have been biochemically characterized as dextranases. Dextran-degrading enzymes have been isolated from a wide range of microorganisms. The major characteristics of these enzymes, the methods for analyzing their activities and biological roles, analysis of primary sequence data, and three-dimensional structures of dextranases have been dealt with in this review. Dextranases are promising for future use in various scientific and biotechnological applications. PMID:15944458

  1. Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts.

    Science.gov (United States)

    Malik Ghulam, Mustafa; Zghidi-Abouzid, Ouafa; Lambert, Emeline; Lerbs-Mache, Silva; Merendino, Livia

    2012-06-01

    The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

  2. Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Cakir, Ayse; Ozmihci, Serpil; Kargi, Fikret [Department of Environmental Engineering, Dokuz Eylul University, Buca, Izmir (Turkey)

    2010-12-15

    Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were compared in batch dark fermentations under mesophilic (37 C) and thermophilic (55 C) conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 C and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H{sub 2} h{sup -1}) was obtained with the acid-hydrolyzed and the lowest (1.12 ml H{sub 2} h{sup -1}) with the boiled wheat at 55 C. The highest hydrogen gas yield (333 ml H{sub 2} g{sup -1} total sugar or 2.40 mol H{sub 2} mol{sup -1} glucose) and final total volatile fatty acid (TVFA) concentration (10.08 g L{sup -1}) were also obtained with the acid-hydrolyzed wheat under thermophilic conditions (55 C). Dark fermentation of acid-hydrolyzed ground wheat under thermophilic conditions (55 C) was proven to be more beneficial as compared to mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch. (author)

  3. High glucose impairs ATP formation on the surface of human peripheral blood B lymphocytes.

    Science.gov (United States)

    Sakowicz-Burkiewicz, Monika; Grden, Marzena; Maciejewska, Izabela; Szutowicz, Andrzej; Pawelczyk, Tadeusz

    2013-07-01

    Diabetes-associated lymphocyte dysfunction may be attributed to the direct effect of hyperglycemia, but the impact of glucose concentration on B cell functionality is not fully resolved. Since, adenosine 5'-triphosphate (ATP) and its metabolite adenosine are the core constituents of the purinergic signaling network involved in regulation of immune response we aimed to investigate the impact of high glucose concentration on ATP outflow and metabolism on B cell surface. Purified human peripheral blood B cells cultured at high glucose (25 mM) concentration released significantly less ATP (~60%) comparing to cells cultured in low glucose (5mM) concentration. We observed that high glucose altered ATP hydrolysis on B cell surface due to increased activity of nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39). In the presence of 10 μM [(3)H]AMP and 100 μM ATP significant quantities of [(3)H]ADP and [(3)H]ATP were generated, although the AMP to ADP phosphorylation potential of B cells cultured in high glucose decreased significantly. The flow cytometry analysis revealed that the level of ecto-adenylate kinase 1β (AK1β) on surface of B cells cultured in high glucose decreased significantly. Inhibition of NTPDase1/CD39 activity with 100 μM ARL67156 resulted in decreased cell viability, although significantly more viable cells retained in the culture media containing low glucose compared to high glucose media. Selective inhibition of P2X7 purinergic receptor irrespective of glucose concentration completely protected B cells against the ARL 67156-induced cell death. We assume that high glucose-induced alteration of ATP handling on B cell surface might contribute to impaired functionality of B cells in diabetes. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.; Sacchettini, James C. (TAM)

    2008-06-23

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

  5. ATP-induced inflammasome activation and pyroptosis is regulated by AMP-activated protein kinase in macrophages

    Directory of Open Access Journals (Sweden)

    Qing-Bing Zha

    2016-12-01

    Full Text Available ATP is released by bacteria and host cells during bacterial infection as well as sterile tissue injury, acting as an inducer of inflammasome activation. Previous studies have shown that ATP treatment leads to AMP-activated protein kinase (AMPK activation. However, it is unclear whether AMPK signaling has been involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis. In this study, we aimed to investigate this issue in lipopolysaccharide-activated murine macrophages. Our results showed that AMPK signaling was activated in murine macrophages upon ATP treatment, which was accompanied by inflammasome activation and pyroptosis as evidenced by rapid cell membrane rupture as well as mature interleukin (IL-1β and active caspase-1p10 release. The ATP-induced inflammasome activation and pyroptosis were markedly suppressed by an AMPK inhibitor compound C or small interfering RNA-mediated knockdown of AMPKα, but could be greatly enhanced by metformin (a well-known AMPK agonist. Importantly, metformin administration increased the mortality of mice with bacterial sepsis, which was likely because metformin treatment enhanced the systemic inflammasome activation as indicated by elevated serum and hepatic IL-1β levels. Collectively, these data indicated that the AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis in macrophages, highlighting the possibility of AMPK-targeting therapies for inflammatory diseases involving inflammasome activation.

  6. Effects of Hydrolyzed Rapeseed Cake Extract on the Quality Characteristics of Mayonnaise Dressing.

    Science.gov (United States)

    Kim, Ye-Seul; Lee, Jeung-Hee

    2017-12-01

    Combined fractions (H2 O and 30% and 50% ethanol) of crude rapeseed cake extracts with 80% ethanol were hydrolyzed with NaOH solution. The hydrolyzed extract showed significantly higher contents of total phenolics (41.8 mg SAE/g) and sinapic acid (425.8 mg/g), as well as higher 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging capacity (91.98 RSC%) than the crude extract (P extract was remarkably higher than that of the crude extract against selected Gram-positive and Gram-negative bacteria as well as yeast, as determined by the minimum inhibitory concentration method. Hydrolyzed extract (100, 250, or 500 ppm) was added to mayonnaise dressing, and several quality characteristics of the dressing were investigated by assessments of microbial, physical, and oxidative stabilities during 8 wk of storage. Microbial stability was higher in the dressing with hydrolyzed extract added (4.3 to 4.6 Log CFU/g) than the control (4.9 Log CFU/g). Physical characteristics of the dressing with hydrolyzed extract added were better than those of the control, based on increased viscosity and reduced emulsion separation. Hydrolyzed extract increased oxidative stability in a concentration-dependent manner, and the dressing with added 500 ppm of hydrolyzed extract resulted in a lower free fatty acid content (4.8% at week 8), peroxide value (13.5 meq/kg at week 6), and 2-thiobarbituric acid-reactive substances value (66.2 μg/100 g at week 8) than the control. Therefore, it is expected that hydrolyzed rapeseed cake extract containing high sinapic acid content can be used in emulsion system as a value-added ingredient. Crude extract of rapeseed cake was fractionated and alkaline-hydrolyzed to convert sinapine into sinapic acid, and the produced hydrolyzed extract showed higher antimicrobial and antioxidative activities than the crude extract. When the hydrolyzed extract was added to mayonnaise dressing, microbial stability increased along with physical characteristics and oxidative

  7. Distinct Conformation of ATP Molecule in Solution and on Protein.

    Science.gov (United States)

    Kobayashi, Eri; Yura, Kei; Nagai, Yoshinori

    2013-01-01

    Adenosine triphosphate (ATP) is a versatile molecule used mainly for energy and a phosphate source. The hydrolysis of γ phosphate initiates the reactions and these reactions almost always start when ATP binds to protein. Therefore, there should be a mechanism to prevent spontaneous hydrolysis reaction and a mechanism to lead ATP to a pure energy source or to a phosphate source. To address these questions, we extensively analyzed the effect of protein to ATP conformation based on the sampling of the ATP solution conformations obtained from molecular dynamics simulation and the sampling of ATP structures bound to protein found in a protein structure database. The comparison revealed mainly the following three points; 1) The ribose ring in ATP molecule, which puckers in many ways in solution, tends to assume either C2' exo or C2' endo when it binds to protein. 2) The adenine ring in ATP molecule, which takes open-book motion with the two ring structures, has two distinct structures when ATP binds to protein. 3) The glycosyl-bond and the bond between phosphate and the ribose have unique torsion angles, when ATP binds to protein. The combination of torsion angles found in protein-bound forms is under-represented in ATP molecule in water. These findings suggest that ATP-binding protein exerts forces on ATP molecule to assume a conformation that is rarely found in solution, and that this conformation change should be a trigger for the reactions on ATP molecule.

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

    Directory of Open Access Journals (Sweden)

    Salam Massadeh

    2014-05-01

    Full Text Available 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 adenosine 5’ triphosphate, glucose, and HEX, a significant photobrightening of the InP/ZnS QDs is recorded. This is dependent on the concentration of ATP in the sample. The relationship between the ATP and the emission intensity of InP/ZnS nanocrystals is linear. The present results are the first to report the effect of different by- products released by these enzymatic reactions on the fluorescence of the InP/ZnS QDs.

  9. 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.

  10. Electric field driven torque in ATP synthase.

    Directory of Open Access Journals (Sweden)

    John H Miller

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

  11. Simultaneous concentration and detoxification of lignocellulosic hydrolyzates by vacuum membrane distillation coupled with adsorption.

    Science.gov (United States)

    Zhang, Yaqin; Li, Ming; Wang, Yafei; Ji, Xiaosheng; Zhang, Lin; Hou, Lian

    2015-12-01

    Low sugar concentration and the presence of various inhibitors are the major challenges associated with lignocellulosic hydrolyzates as a fermentation broth. Vacuum membrane distillation (VMD) process can be used to concentrate sugars and remove inhibitors (furans) efficiently, but it's not desirable for the removal of less volatile inhibitors such as acetic acid. In this study, a VMD-adsorption process was proposed to improve the removal of acetic acid, achieving simultaneous concentration and detoxification of lignocellulosic hydrolyzates by one step process. Results showed that sugars were concentrated with high rejections (>98%) and little sugar loss (<2%), with the significant reduction in nearly total furans (99.7%) and acetic acid (83.5%) under optimal operation conditions. Fermentation results showed the ethanol production of hydrolyzates concentrated and detoxified using the VMD-adsorption method were approximately 10-fold greater than from untreated hydrolyzates. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Hydrolyzed casein reduces diet-induced obesity in male C57BL/6J mice

    DEFF Research Database (Denmark)

    Lillefosse, Haldis Haukås; Tastesen, Hanne Sørup; Du, Zhen-Yu

    2013-01-01

    The digestion rate of dietary protein is a regulating factor for postprandial metabolism both in humans and animal models. However, few data exist about the habitual consumption of proteins with different digestion rates with regard to the development of body mass and diet-induced obesity. Here, we...... used a factorial ANOVA design to investigate the effects of protein form (intact vs. hydrolyzed casein) and protein level (16 vs. 32 energy percent protein) on body mass gain and adiposity in obesity-prone male C57BL/6J mice fed Western diets with 35 energy percent fat. Mice fed the hydrolyzed casein...... by hydrolyzed casein ingestion translated into decreased body and adipose tissue masses. We conclude that chronic consumption of extensively hydrolyzed casein reduces body mass gain and diet-induced obesity in male C57BL/6J mice....

  13. Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

    Energy Technology Data Exchange (ETDEWEB)

    Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

    2016-04-26

    The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

  14. Recombinant, rice-produced yeast phytase shows the ability to hydrolyze phytate derived from seed-based feed, and extreme stability during ensilage treatment.

    Science.gov (United States)

    Hamada, Akira; Yamaguchi, Ken-Ichi; Harada, Michiko; Horiguchi, Ken-Ichi; Takahashi, Toshiyoshi; Honda, Hideo

    2006-06-01

    When fresh rice leaves producing yeast Schwanniomyces occidentalis phytase were grounded and mixed with the whole extract of seed-based feed for pigs, the release of orthophosphate increased significantly. More specifically, phytate, a major source of phosphorus in the seeds, was hydrolyzed by heterologous phytase. Moreover, when transgenic rice plants were ensiled for up to 12 weeks, no decrease in the phytase activity of the heterologous enzyme was observed. This result strongly suggests that transgenic rice plants producing yeast phytase can be stored as silage without any loss of enzyme activity until usage as a feed additive.

  15. The relationship between absorbency and density of bioplastic film made from hydrolyzed starch

    Science.gov (United States)

    Singan, Grace; Chiang, Liew Kang

    2017-12-01

    Water absorption in polymer blends such as starch-based bioplastic films is important to evaluate the stability characteristics of such films in water that will affect their long-term performance in final products. In this study, the absorbency of starch-based bioplastic films made from potato, cassava, and corn starches that have went through the hydrolysis process first to alter its characteristics and properties in terms of granular swelling and hydrophilicity behaviour. The final results showed that hydrolyzed cassava bioplastic film has the ability to absorb more water compared to hydrolyzed potato and corn bioplastic films. The reading of hydrolyzed cassava bioplastic film on the seventh day of immersion for all ratios were between 87.83 % to 131.29 %, while for hydrolyzed potato bioplastic films was 69.48 % to 92.41 % and hydrolyzed corn bioplastic films was 66.28 % to 74.18 %. Meanwhile, the density analysis was evaluated to determine its physical properties towards moisture condition. The results showed that the hydrolyzed cassava bioplastic films have higher density compared to the other two, which indicated that it is a more favourable raw material to produce biodegradable planting pot due to its ability to absorb more water. Hence, still manage to retain its shape with low brittle surface.

  16. Antimicrobial activity of poultry bone and meat trimmings hydrolyzates in low-sodium turkey food.

    Science.gov (United States)

    Zanello, Pier Paolo; Sforza, Stefano; Dossena, Arnaldo; Lambertini, Francesca; Bottesini, Chiara; Nikolaev, Ilya V; Koroleva, Olga; Ciociola, Tecla; Magliani, Walter; Conti, Stefania; Polonelli, Luciano

    2014-02-01

    This research was aimed at the evaluation of the antimicrobial activity exerted by poultry protein hydrolyzates derived from industrial leftovers added to minced turkey meat, intended for the production of burgers for human consumption. Hydrolyzates were obtained through enzymatic hydrolysis from poultry bone and meat trimmings, as by-products from the poultry industry. Colony forming unit assays, under both laboratory and industrial conditions, were performed to assess microbial growth. Poultry protein hydrolyzates inhibited microbial growth occurring in semi-finished turkey meat during the normal retention period because of their water holding capacity resulting in a decreased water activity. Overall, the findings demonstrated that poultry protein hydrolyzates could decrease mesophilic, psychrophilic, and thermophilic bacterial growth for the entire product shelf-life. Bacterial growth inhibition obtained in minced turkey meat by addition of poultry protein hydrolyzates (1.5%), hygroscopic amino acids mixture (1.5%) or sodium chloride (1%) was similar. It is suggested that the use of hydrolyzates could allow the reduction of salt content in poultry meat based products leading to the production of low-sodium turkey food still maintaining acceptable sensory characteristics.

  17. Isothermal amplified detection of ATP using Au nanocages capped with a DNA molecular gate and its application in cell lysates.

    Science.gov (United States)

    Wang, Wei; Zhao, Na; Li, Xiaoxiao; Wan, Jun; Luo, Xiliang

    2015-03-07

    A novel controlled-release biosensor for isothermal amplified detection of ATP using Au nanocages (AuNCs) capped with a DNA molecular gate is reported for the first time, and has been successfully tested in intracellular ATP detection. Two kinds of SH-modified short strand DNAs S1 and S2 were assembled on the surface of the AuNCs by means of Au-thiolate bonding. The hybridization of a long-strand DNA S3 with the two immobilized SH-DNAs leads to the formation of molecular gates. The molecular gates were designed to inhibit the release of the fluorescent molecules such as Rhodamine-B (RhB), which were filled in the hollow interiors of AuNCs. The primer S4 was employed to play the role of a recognition moiety. The specificity recognition reaction between ATP and ATP aptamer gave rise to the primer S4 released from a double-stranded hybrid formed with the ATP aptamer. The released S4 will initiate the autonomous replication-scission-displacement process with the assistance of DNA polymerase and nicking endonuclease. As a result, the DNA synthesis and the DNA cycle achieved the opening of the DNA-based molecular gates and the significant amplification of the release of the guest molecules from AuNCs. In order to realize the cyclic enzymatic amplification of the release of the guest molecules from AuNCs, the long-strand S3 is ingeniously designed in such a way that it contains a Nb.Bpu10I nicking endonuclease recognition sequence and a sequence complementary to the primer S4. The fabricated system was demonstrated to be an efficient biosensor for target molecule detection qualitatively and quantitatively.

  18. 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

  19. Limitations of ATP as a measure of microbial biomass | Stuart ...

    African Journals Online (AJOL)

    Estimates of the total living biomass of micro-organisms on decomposing kelp detritus, calculated indirectly from the concentration of ATP, were compared with those obtained directly from cell numbers and volumes. Large overestimates in biomass were obtained from ATP x 250, and C:ATP ratios varied considerably with ...

  20. 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.

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

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast...

  2. 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

  3. Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking.

    Science.gov (United States)

    Yi, Ling; Kaler, Stephen G

    2015-05-01

    ATP7A is a P-type ATPase in which diverse mutations lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two previously unknown ATP7A missense mutations, T994I and P1386S, were shown to cause an isolated distal motor neuropathy without clinical or biochemical features of other ATP7A disorders. These mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma membrane localization compared with wild-type ATP7A. We reported previously that ATP7A(P1386S) causes unstable insertion of the eighth and final transmembrane segment, preventing proper position of the carboxyl-terminal tail in a proportion of mutant molecules. Here, we utilize this and other naturally occurring and engineered mutant ATP7A alleles to identify mechanisms of normal ATP7A trafficking. We show that adaptor protein (AP) complexes 1 and 2 physically interact with ATP7A and that binding is mediated in part by a carboxyl-terminal di-leucine motif. In contrast to other ATP7A missense mutations, ATP7A(P1386S) partially disturbs interactions with both APs, leading to abnormal axonal localization in transfected NSC-34 motor neurons and altered calcium-signaling following glutamate stimulation. Our results imply that AP-1 normally tethers ATP7A at the trans-Golgi network in the somatodendritic segments of motor neurons and that alterations affecting the ATP7A carboxyl-terminal tail induce release of the copper transporter to the axons or axonal membranes. The latter effects are intensified by diminished interaction with AP-2, impeding ATP7A retrograde trafficking. Taken together, these findings further illuminate the normal molecular mechanisms of ATP7A trafficking and suggest a pathophysiological basis for ATP7A-related distal motor neuropathy. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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

    Science.gov (United States)

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

    1974-01-01

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

  5. ATP Hydrolysis Induced Conformational Changes in the Vitamin B12 Transporter BtuCD Revealed by MD Simulations.

    Science.gov (United States)

    Pan, Chao; Weng, Jingwei; Wang, Wenning

    2016-01-01

    ATP binding cassette (ABC) transporters utilize the energy of ATP hydrolysis to uni-directionally transport substrates across cell membrane. ATP hydrolysis occurs at the nucleotide-binding domain (NBD) dimer interface of ABC transporters, whereas substrate translocation takes place at the translocation pathway between the transmembrane domains (TMDs), which is more than 30 angstroms away from the NBD dimer interface. This raises the question of how the hydrolysis energy released at NBDs is "transmitted" to trigger the conformational changes at TMDs. Using molecular dynamics (MD) simulations, we studied the post-hydrolysis state of the vitamin B12 importer BtuCD. Totally 3-μs MD trajectories demonstrate a predominantly asymmetric arrangement of the NBD dimer interface, with the ADP-bound site disrupted and the ATP-bound site preserved in most of the trajectories. TMDs response to ATP hydrolysis by separation of the L-loops and opening of the cytoplasmic gate II, indicating that hydrolysis of one ATP could facilitate substrate translocation by opening the cytoplasmic end of translocation pathway. It was also found that motions of the L-loops and the cytoplasmic gate II are coupled with each other through a contiguous interaction network involving a conserved Asn83 on the extended stretch preceding TM3 helix plus the cytoplasmic end of TM2/6/7 helix bundle. These findings entail a TMD-NBD communication mechanism for type II ABC importers.

  6. Human MSH2 (hMSH2) protein controls ATP processing by hMSH2-hMSH6.

    Science.gov (United States)

    Heinen, Christopher D; Cyr, Jennifer L; Cook, Christopher; Punja, Nidhi; Sakato, Miho; Forties, Robert A; Lopez, Juana Martin; Hingorani, Manju M; Fishel, Richard

    2011-11-18

    The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ∼8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.

  7. Role of lipase from community-associated methicillin-resistant Staphylococcus aureus strain USA300 in hydrolyzing triglycerides into growth-inhibitory free fatty acids.

    Science.gov (United States)

    Cadieux, Brigitte; Vijayakumaran, Vithooshan; Bernards, Mark A; McGavin, Martin J; Heinrichs, David E

    2014-12-01

    Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

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

    2014-06-01

    Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. 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.

  10. Properties of Na+ ,K+ -activated, Mg2+ -dependent ATP-hydrolyze of blood lymphocytes in patients with reаctive arthritis

    Directory of Open Access Journals (Sweden)

    O.V. Melnyk

    2013-11-01

    with RеA. We observed that Na+,K+-ATPase of peripheral blood lymphocytes of patients with RеA retains its endogenous receptor properties – sensitivity to ouabain does not change. It is assumed that under conditions of rheumatic pathology the impact on the Na+,K+-ATPase structure occurs both externally and on the cytoplasmic membrane surface. The above experimental data can be used for further clarification of the membrane mechanisms of ion exchange in immunocompetent cells of patients suffering from autoimmune diseases.

  11. A Vision of Interdisciplinary Education: Students' Reasoning about "High-Energy Bonds" and ATP

    CERN Document Server

    Dreyfus, Benjamin W; Turpen, Chandra; Gouvea, Julia; Redish, Edward F

    2014-01-01

    As interdisciplinary courses are developed, instructors and researchers have to grapple with questions of how students should make connections across disciplines. We explore the issue of interdisciplinary reconciliation (IDR): how students reconcile seemingly contradictory ideas from different disciplines. While IDR has elements in common with other frameworks for the reconciliation of ideas across contexts, it differs in that each disciplinary idea is considered canonically correct within its own discipline. The setting for the research is an introductory physics course for biology majors that seeks to build greater interdisciplinary coherence and therefore includes biologically relevant topics such as ATP and chemical bond energy. In our case-study data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see students justifying context-dependent modeling choices, showing nuanc...

  12. Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin.

    Science.gov (United States)

    Takahashi, Y; Mitsui, A; Fujita, Y; Matsubara, H

    1991-01-01

    Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [(35)S]cysteine, in which [(35)S]sulfide and iron are incorporated into Fd to build up the (35)S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [(35)S]sulfide or iron, or in Fe-S cluster formation itself. [(35)S]Sulfide was liberated from [(35)S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [(35)S]cysteine occurred before the formation of the (35)S-labeled Fe-S cluster, and the amount of radioactivity in [(35)S]sulfide was greater than that in (35)S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na(2)S) inhibited competitively formation of the (35)S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [(35)S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster

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

    Directory of Open Access Journals (Sweden)

    Emri Zsuzsa

    2011-10-01

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

  14. Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca2+ signals and an IL-6 autocrine loop

    Science.gov (United States)

    Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique

    2014-01-01

    Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop. PMID:24518675

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

    Directory of Open Access Journals (Sweden)

    Ting Xu

    2015-03-01

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

  16. MRT letter: Expression of ATP sensor protein in Caenorhabditis elegans.

    Science.gov (United States)

    Kishikawa, Jun-ichi; Fujikawa, Makoto; Imamura, Hiromi; Yasuda, Kayo; Noji, Hiroyuki; Ishii, Naoaki; Mitani, Shohei; Yokoyama, Ken

    2012-01-01

    Adenosine 5'-triphosphate (ATP) is the major energy currency and is involved in many biological processes. The ATP-monitoring system for cells in animals can be helpful to study the relationship between energy metabolism and biological processes. The fluorescent ATP biosensor ATeam (ATP indicator based on Epsilon subunit for Analytical Measurements), which has been reported to monitor ATP levels in cultured cells on the basis of fluorescence resonance energy transfer (FRET), was introduced into nematodes by microinjection and UV-irradiation method. To confirm whether ATeam functions as an ATP sensor in nematode cells, the authors measured FRET of ATeam in cells of transgenic nematode. The ATeam was expressed in target cells in nematode. In vulva cells, ATP levels in the cytosol were higher than those in mitochondria. ATeam also sensed ATP level change in cultured cells from the transgenic nematode. These experiments indicated that ATeam is available for detection of changes in ATP levels in nematode cells. Copyright © 2011 Wiley Periodicals, Inc.

  17. Formation of a raw starch-hydrolyzing -amlyase by Clostridium 2021: effect of carbon sources

    Energy Technology Data Exchange (ETDEWEB)

    Avendano, M.C.; Cornejo, I.

    1987-01-01

    Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3% concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose, glucose and higher dextrins.

  18. Toughness of natural rubber composites reinforced with hydrolyzed and modified wheat gluten aggregates

    Science.gov (United States)

    The toughness of natural rubber can be improved by using fillers for various rubber applications. Dry wheat gluten is a protein from wheat flour and is sufficiently rigid for rubber reinforcement. The wheat gluten was hydrolyzed to reduce its particle size and microfluidized to reduce and homogenize...

  19. Effect of partially hydrolyzed guar gum (PHGG) on the bioaccessibility of fat and cholesterol

    NARCIS (Netherlands)

    Minekus, M.; Jelier, M.; Xiao, J.-Z.; Kondo, S.; Iwatsuki, K.; Kokubo, S.; Bos, M.; Dunnewind, B.; Havenaar, R.

    2005-01-01

    The Addition of a compound that lowers the intestinal uptake of fat and cholesterol might be an interesting strategy to reduce the risk of vascular disease. Partially hydrolyzed guar gum (PHGG) has been shown to have this effect in healthy volunteers after intake of a yogurt drink with 3 to 6% PHGG.

  20. Rheology of dilute acid hydrolyzed corn stover at high solids concentration

    Science.gov (United States)

    M.R. Ehrhardt; T.O. Monz; T.W. Root; R.K. Connelly; Tim Scott; D.J. Klingenberg

    2010-01-01

    The rheological properties of acid hydrolyzed corn stover at high solids concentration (20–35 wt.%) were investigated using torque rheometry. These materials are yield stress fluids whose rheological properties can be well represented by the Bingham model. Yield stresses increase with increasing solids concentration and decrease with increasing hydrolysis reaction...

  1. Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Neelamegan Dhamodharan

    2012-06-01

    Full Text Available Abstract Background Anandamide (Arachidonoyl ethanolamide is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH. In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide. Results A putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates. Conclusions This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.

  2. Fermentation of dilute acid hydrolyzates. Effects of mode of operation and medium composition

    Energy Technology Data Exchange (ETDEWEB)

    Liden, G. [Chalmers Univ. of Technology, Goeteborg (Sweden). Dept. of Chemical Reaction Engineering]|[Goeteborg Univ. (Sweden). Dept. of General and Marine Microbiology

    1997-11-01

    The fermentation of dilute acid hydrolyzates is hampered by the presence of inhibitors in the hydrolyzates. In most design studies it is assumed that these inhibitors have to be removed before fermentation. In the present work an alternative to detoxification, fed-batch operation is studied. The fed-batch process is based on uptake of furfural and HMF from the medium, and for this reason the uptake of these compounds was studied also independently. A strongly inhibiting hydrolyzate of spruce was then used as a test medium for a fed-batch process, and different feed-rates were examined. It was indeed found possible to ferment the strongly inhibiting substrate without prior detoxification using a fed-batch technique. However, the fermentation rate is limited, most probably by the uptake rate of furfural and HMF from the hydrolyzates. As shown by the uptake study, the physiological status of the cells has a large influence on the uptake of furfural. This, in turn, will decide maximum feed rates for the fed-batch process 3 refs, 13 figs, 1 tab

  3. Immunologic responses against hydrolyzed soy protein in dogs with experimentally induced soy hypersensitivity.

    Science.gov (United States)

    Puigdemont, Anna; Brazís, Pilar; Serra, Montserrat; Fondati, Alessandra

    2006-03-01

    To assess whether dogs with experimentally induced type I hypersensitivity against soy protein would respond to soy hydrolysate and develop cutaneous or gastrointestinal tract reactions after intradermal and oral challenge exposure. 12 naïve Beagle pups (9 sensitized and 3 control dogs). 9 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization period, serum concentrations of soy-specific IgE were determined and an intradermal test was performed to confirm the dogs were sensitized against soy protein. An intradermal challenge test and an oral challenge test with native and hydrolyzed soy protein were conducted on 6 sensitized and 2 control dogs. High serum concentrations of soy-specific IgE and positive results for the intradermal test were observed for the 9 sensitized dogs after completion of the sesitization process. Sensitized dogs challenge exposed with hydrolyzed soy protein had a reduced inflammatory response after intradermal injection and no clinical response after an oral challenge exposure, compared with responses after intradermal and oral challenge exposure with native soy protein. Soy-sensitized dogs did not respond to oral administration of hydrolyzed soy protein. Thus, hydrolyzed soy protein may be useful in diets formulated for the management of dogs with adverse reactions to food.

  4. Kinetic characteristics of polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry

    Science.gov (United States)

    Polygalacturonase enzymes hydrolyze the polygalacturonic acid chains found in pectin. Interest in polygalacturonase enzymes continues as they are useful in a number of industrial processes and conversely, detrimental, as they are involved in maceration of economically important crops. While a good...

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

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    Zhengchang Liu

    2013-03-01

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

  6. Adenosine-5'-triphosphate (ATP supplementation improves low peak muscle torque and torque fatigue during repeated high intensity exercise sets

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    Rathmacher John A

    2012-10-01

    Full Text Available Abstract Background Intracellular concentrations of adenosine-5’-triphosphate (ATP are many times greater than extracellular concentrations (1–10 mM versus 10–100 nM, respectively and cellular release of ATP is tightly controlled. Transient rises in extracellular ATP and its metabolite adenosine have important signaling roles; and acting through purinergic receptors, can increase blood flow and oxygenation of tissues; and act as neurotransmitters. Increased blood flow not only increases substrate availability but may also aid in recovery through removal of metabolic waste products allowing muscles to accomplish more work with less fatigue. The objective of the present study was to determine if supplemental ATP would improve muscle torque, power, work, or fatigue during repeated bouts of high intensity resistance exercise. Methods Sixteen participants (8 male and 8 female; ages: 21–34 years were enrolled in a double-blinded, placebo-controlled study using a crossover design. The participants received either supplemental ATP (400 mg/d divided into 2 daily doses or placebo for 15 d. After an overnight fast, participants underwent strength and fatigue testing, consisting of 3 sets of 50 maximal knee extensions performed on a Biodex® leg dynamometer. Results No differences were detected in high peak torque, power, or total work with ATP supplementation; however, low peak torque in set 2 was significantly improved (p Conclusions Supplementation with 400 mg ATP/d for 15 days tended to reduce muscle fatigue and improved a participant’s ability to maintain a higher force output at the end of an exhaustive exercise bout.

  7. Modelling the ATP production in mitochondria

    CERN Document Server

    Saa, Alberto

    2012-01-01

    We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer's model. We correct some inaccuracies in the BPLS original approximations and then analyze some of the dynamical properties of the model. We infer from exhaustive numerical explorations that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium ${\\rm Ca}_{\\rm c}$ and the substrate fructose 1,6-bisphosphate FBP. The same effect of calcium saturation reported for the original BPLS model is observed here. We find out, however, an interesting non-stationary effect: the inertia of the model tends to increase considerably for high concentrations of ...

  8. Selected Probiotic Lactobacilli Have the Capacity To Hydrolyze Gluten Peptides during Simulated Gastrointestinal Digestion.

    Science.gov (United States)

    Francavilla, Ruggiero; De Angelis, Maria; Rizzello, Carlo Giuseppe; Cavallo, Noemi; Dal Bello, Fabio; Gobbetti, Marco

    2017-07-15

    The aim of this study was to demonstrate the capacity of probiotic lactobacilli to hydrolyze immunogenic gluten peptides. Eighteen commercial strains of probiotic lactobacilli with highly variable peptidase activity (i.e., aminopeptidase N, iminopeptidase, prolyl endopeptidyl peptidase, tripeptidase, prolidase, prolinase, and dipeptidase), including toward Pro-rich peptides, were tested in this study. Ten probiotic strains were selected on the basis of their specific enzyme activity. When pooled, these 10 strains provided the peptidase portfolio that is required to completely degrade the immunogenic gluten peptides involved in celiac disease (CD). The selected probiotic mixture was able to completely hydrolyze well-known immunogenic epitopes, including the gliadin 33-mer peptide, the peptide spanning residues 57 to 68 of the α9-gliadin (α9-gliadin peptide 57-68), A-gliadin peptide 62-75, and γ-gliadin peptide 62-75. During digestion under simulated gastrointestinal conditions, the pool of 10 selected probiotic lactobacilli strongly hydrolyzed the wheat bread gluten (ca. 18,000 ppm) to less than 10 ppm after 360 min of treatment. As determined by multidimensional chromatography (MDLC) coupled to nanoelectrospray ionization (nano-ESI)-tandem mass spectrometry (MS/MS), no known immunogenic peptides were detected in wheat bread that was digested in the presence of the probiotics. Accordingly, the level of cytokines (interleukin 2 [IL-2], IL-10, and interferon gamma [IFN-γ]) produced by duodenal biopsy specimens from CD patients who consumed wheat bread digested by probiotics was similar to the baseline value (negative control). Probiotics that specifically hydrolyze gluten polypeptides could also be used to hydrolyze immunogenic peptides that contaminate gluten-free products. This could provide a new and safe adjunctive therapy alternative to the gluten-free diet (GFD). IMPORTANCE This study confirmed that probiotic Lactobacillus strains have different enzymatic

  9. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.

    Science.gov (United States)

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D

    2015-12-15

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Directory of Open Access Journals (Sweden)

    Bernd L. Fiebich

    2014-09-01

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

  11. Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

    Science.gov (United States)

    Yu, Corey H; Dolgova, Natalia V; Dmitriev, Oleg Y

    2017-04-01

    Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  12. Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials

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    Murakami Katsuji

    2009-10-01

    Full Text Available Abstract Background Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi. Results We compared A. cellulolyticus and T. reesei cellulase activity against the three lignocellulosic materials: eucalyptus, Douglas fir and rice straw. Saccharification analysis using the supernatant from each culture demonstrated that the enzyme mixture derived from A. cellulolyticus exhibited 2-fold and 16-fold increases in Filter Paper enzyme and β-glucosidase specific activities, respectively, compared with that derived from T. reesei. In addition, culture supernatant from A. cellulolyticus produced glucose more rapidly from the lignocellulosic materials. Meanwhile, culture supernatant derived from T. reesei exhibited a 2-fold higher xylan-hydrolyzing activity and produced more xylose from eucalyptus (72% yield and rice straw (43% yield. Although the commercial enzymes Acremonium cellulase (derived from A. cellulolyticus, Meiji Seika Co. demonstrated a slightly lower cellulase specific activity than Accellerase 1000 (derived from T. reesei, Genencor, the glucose yield (over 65% from lignocellulosic materials by Acremonium cellulase was higher than that of Accellerase 1000 (less than 60%. In addition, the mannan-hydrolyzing activity of Acremonium cellulase was 16-fold higher than that of Accellerase 1000, and the conversion of mannan to mannobiose and mannose by Acremonium cellulase was more efficient. Conclusion We investigated the hydrolysis of lignocellulosic materials by cellulase derived from two types of filamentous fungi. We

  13. Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials.

    Science.gov (United States)

    Fujii, Tatsuya; Fang, Xu; Inoue, Hiroyuki; Murakami, Katsuji; Sawayama, Shigeki

    2009-10-01

    Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi. We compared A. cellulolyticus and T. reesei cellulase activity against the three lignocellulosic materials: eucalyptus, Douglas fir and rice straw. Saccharification analysis using the supernatant from each culture demonstrated that the enzyme mixture derived from A. cellulolyticus exhibited 2-fold and 16-fold increases in Filter Paper enzyme and beta-glucosidase specific activities, respectively, compared with that derived from T. reesei. In addition, culture supernatant from A. cellulolyticus produced glucose more rapidly from the lignocellulosic materials. Meanwhile, culture supernatant derived from T. reesei exhibited a 2-fold higher xylan-hydrolyzing activity and produced more xylose from eucalyptus (72% yield) and rice straw (43% yield). Although the commercial enzymes Acremonium cellulase (derived from A. cellulolyticus, Meiji Seika Co.) demonstrated a slightly lower cellulase specific activity than Accellerase 1000 (derived from T. reesei, Genencor), the glucose yield (over 65%) from lignocellulosic materials by Acremonium cellulase was higher than that of Accellerase 1000 (less than 60%). In addition, the mannan-hydrolyzing activity of Acremonium cellulase was 16-fold higher than that of Accellerase 1000, and the conversion of mannan to mannobiose and mannose by Acremonium cellulase was more efficient. We investigated the hydrolysis of lignocellulosic materials by cellulase derived from two types of filamentous fungi. We found that glucan-hydrolyzing activity of the culture

  14. Glucose elicits cephalic-phase insulin release in mice by activating KATP channels in taste cells.

    Science.gov (United States)

    Glendinning, John I; Frim, Yonina G; Hochman, Ayelet; Lubitz, Gabrielle S; Basile, Anthony J; Sclafani, Anthony

    2017-04-01

    The taste of sugar elicits cephalic-phase insulin release (CPIR), which limits the rise in blood glucose associated with meals. Little is known, however, about the gustatory mechanisms that trigger CPIR. We asked whether oral stimulation with any of the following taste stimuli elicited CPIR in mice: glucose, sucrose, maltose, fructose, Polycose, saccharin, sucralose, AceK, SC45647, or a nonmetabolizable sugar analog. The only taste stimuli that elicited CPIR were glucose and the glucose-containing saccharides (sucrose, maltose, Polycose). When we mixed an α-glucosidase inhibitor (acarbose) with the latter three saccharides, the mice no longer exhibited CPIR. This revealed that the carbohydrates were hydrolyzed in the mouth, and that the liberated glucose triggered CPIR. We also found that increasing the intensity or duration of oral glucose stimulation caused a corresponding increase in CPIR magnitude. To identify the components of the glucose-specific taste-signaling pathway, we examined the necessity of Calhm1, P2X2+P2X3, SGLT1, and Sur1. Among these proteins, only Sur1 was necessary for CPIR. Sur1 was not necessary, however, for taste-mediated attraction to sugars. Given that Sur1 is a subunit of the ATP-sensitive K+ channel (KATP) channel and that this channel functions as a part of a glucose-sensing pathway in pancreatic β-cells, we asked whether the KATP channel serves an analogous role in taste cells. We discovered that oral stimulation with drugs known to increase (glyburide) or decrease (diazoxide) KATP signaling produced corresponding changes in glucose-stimulated CPIR. We propose that the KATP channel is part of a novel signaling pathway in taste cells that mediates glucose-induced CPIR. Copyright © 2017 the American Physiological Society.

  15. Exon duplications in the ATP7A gene

    DEFF Research Database (Denmark)

    Mogensen, Mie; Skjørringe, Tina; Kodama, Hiroko

    2011-01-01

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

  16. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  17. Expression of ATP7B in normal human liver

    Directory of Open Access Journals (Sweden)

    D Fanni

    2009-06-01

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

  18. Nanomolar ambient ATP decelerates P2X3 receptor kinetics.

    Science.gov (United States)

    Grote, Alexander; Hans, Michael; Boldogkoi, Zsolt; Zimmer, Andreas; Steinhäuser, Christian; Jabs, Ronald

    2008-12-01

    Homomeric P2X receptors differ in their electrophysiological and pharmacological profiles. The rapidly activating and desensitizing P2X3 receptors are known for their involvement in pain signalling pathways. Modulatory effects on P2X3 receptors have been reported for low concentrations of ATP ([ATP]). This includes both, enhancement and reduction of receptor currents. The first has been reported to be mediated by activation of ectoprotein kinases and high affinity desensitization (HAD), respectively. Both processes influence receptor current amplitudes. Here we describe a new phenomenon, the modulatory influence of ambient low [ATP] on P2X3 receptor kinetics. First, we studied in HEK cells whether persistent ATP affects current decay. To this end, P2X3 receptor mediated currents, elicited by pressure application of saturating [ATP], were analyzed after pre-application of low [ATP]. Second, UV-flash photolysis of ATP was employed to investigate whether submicromolar [ATP] affects receptor activation. Finally we confirmed the action of nanomolar [ATP] on native P2X3 receptors of neurons freshly isolated from rat dorsal root ganglia. We found that persistent low [ATP] caused pronounced deceleration of receptor current activation and decay. This priming effect indicates a mechanism different from HAD. It could be explained by a pre-opening receptor isomerization, induced by the occupation of a high affinity binding site already at the resting state. The observed modulation of the receptor kinetics could be considered as a physiological fine tuning mechanism of the nociceptive system, driven by the actual ambient agonist concentration.

  19. Characterization of causative allergens for wheat-dependent exercise-induced anaphylaxis sensitized with hydrolyzed wheat proteins in facial soap.

    Science.gov (United States)

    Yokooji, Tomoharu; Kurihara, Saki; Murakami, Tomoko; Chinuki, Yuko; Takahashi, Hitoshi; Morita, Eishin; Harada, Susumu; Ishii, Kaori; Hiragun, Makiko; Hide, Michihiro; Matsuo, Hiroaki

    2013-12-01

    In Japan, hydrolyzed wheat proteins (HWP) have been reported to cause wheat-dependent exercise-induced anaphylaxis (WDEIA) by transcutaneous sensitization using HWP-containing soap. Patients develop allergic reactions not only with soap use, but also with exercise after the intake of wheat protein (WP). ω5-Gliadin and HMW-glutenin were identified as major allergens in conventional WP-WDEIA patients. However, the allergens in HWP-WDEIA have yet to be elucidated. Sera were obtained from 22 patients with HWP-sensitized WDEIA. The allergenic activities of HWP and six recombinant wheat gluten proteins, including α/β-, γ-, ω1,2- and ω5-gliadin and low- and high molecular weight (HMW)-glutenins, were characterized by immunoblot analysis and histamine releasing test. IgE-binding epitopes were identified using arrays of overlapping peptides synthesized on SPOTs membrane. Immunoblot analysis showed that IgE antibodies (Abs) from HWP-WDEIA bound to α/β-, γ- and ω1,2-gliadin. Recombinant γ-gliadin induced significant histamine release from basophils in eight of 11 patients with HWP-WDEIA. An IgE-binding epitope "QPQQPFPQ" was identified within the primary sequence of γ-gliadin, and the deamidated peptide containing the "PEEPFP" sequence bound with IgE Abs more strongly compared to the native epitope-peptide. The epitope-peptide inhibited IgE-binding to HWP, indicating that the specific IgE to HWP cross-reacts with γ-gliadin. HWP-WDEIA patients could be sensitized to HWP containing a PEEPFP sequence, and WDEIA symptoms after WP ingestion could partly be induced by γ-gliadin. These findings could be useful to help develop tools for diagnosis and desensitization therapy for HWP-WDEIA.

  20. 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.

  1. 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

  2. Biowaiver Monographs for Immediate-Release Solid Oral Dosage Forms: Enalapril.

    NARCIS (Netherlands)

    Verbeeck, Roger K; Kanfer, Isadore; Löbenberg, Raimar; Abrahamsson, Bertil; Cristofoletti, Rodrigo; Groot, D W; Langguth, Peter; Polli, James E; Parr, Alan; Shah, Vinod P; Mehta, Mehul; Dressman, Jennifer B

    Literature data relevant to the decision to allow a waiver of in vivo bioequivalence testing for the marketing authorization of immediate-release, solid oral dosage forms containing enalapril maleate are reviewed. Enalapril, a prodrug, is hydrolyzed by carboxylesterases to the active

  3. Vapor-phase diethyl oxalate pretreatment of wood chips. Part 2, Release of hemicellulosic carbohydrates

    Science.gov (United States)

    William Kenealy; Eric Horn; Mark Davis; Ross Swaney; Carl Houtman

    2007-01-01

    Wood chips of pine, spruce, aspen, and maple were treated at 135–1408C with diethyl oxalate (DEO) and analyzed for extractable and residual carbohydrates. Under these conditions, DEO hydrolyzes to ethanol and oxalic acid (OA). The amount and identity of carbohydrates released from the chips were species-dependent. For all wood species, increasing the amount of chemical...

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

    Science.gov (United States)

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

    2016-05-01

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

  5. In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    OpenAIRE

    Wagner, Stefan; Chiosea, Simion; Ivshina, Maria; Nickerson, Jeffrey A.

    2004-01-01

    We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splic...

  6. What Orthopaedic Operating Room Surfaces Are Contaminated With Bioburden? A Study Using the ATP Bioluminescence Assay.

    Science.gov (United States)

    Richard, Raveesh Daniel; Bowen, Thomas R

    2017-07-01

    Contaminated operating room surfaces can increase the risk of orthopaedic infections, particularly after procedures in which hardware implantation and instrumentation are used. The question arises as to how surgeons can measure surface cleanliness to detect increased levels of bioburden. This study aims to highlight the utility of adenosine triphosphate (ATP) bioluminescence technology as a novel technique in detecting the degree of contamination within the sterile operating room environment. What orthopaedic operating room surfaces are contaminated with bioburden? When energy is required for cellular work, ATP breaks down into adenosine biphosphate (ADP) and phosphate (P) and in that process releases energy. This process is inherent to all living things and can be detected as light emission with the use of bioluminescence assays. On a given day, six different orthopaedic surgery operating rooms (two adult reconstruction, two trauma, two spine) were tested before surgery with an ATP bioluminescence assay kit. All of the cases were considered clean surgery without infection, and this included the previously performed cases in each sampled room. These rooms had been cleaned and prepped for surgery but the patients had not been physically brought into the room. A total of 13 different surfaces were sampled once in each room: the operating room (OR) preparation table (both pre- and postdraping), OR light handles, Bovie machine buttons, supply closet countertops, the inside of the Bair Hugger™ hose, Bair Hugger™ buttons, right side of the OR table headboard, tourniquet machine buttons, the Clark-socket attachment, and patient positioners used for total hip and spine positioning. The relative light units (RLUs) obtained from each sample were recorded and data were compiled and averaged for analysis. These values were compared with previously published ATP benchmark values of 250 to 500 RLUs to define cleanliness in both the hospital and restaurant industries. All

  7. EndoSd: an IgG glycan hydrolyzing enzyme in Streptococcus dysgalactiae subspecies dysgalactiae.

    Science.gov (United States)

    Shadnezhad, Azadeh; Naegeli, Andreas; Sjögren, Jonathan; Adamczyk, Barbara; Leo, Fredrik; Allhorn, Maria; Karlsson, Niclas G; Jensen, Anders; Collin, Mattias

    2016-06-01

    The aim of this study was to identify and characterize EndoS-like enzymes in Streptococcus dysgalactiae subspecies dysgalactiae (SDSD). PCR, DNA sequencing, recombinant protein expression, lectin blot, ultra high performance liquid chromatography analysis and a chitinase assay were used to identify ndoS-like genes and characterize EndoSd. EndoSd were found in four SDSD strains. EndoSd hydrolyzes the chitobiose core of the glycan on IgG. The amino acid sequence of EndoSd is 70% identical to EndoS in S. pyogenes, but it has a unique C-terminal sequence. EndoSd secretion is influenced by the carbohydrate composition of the growth medium. Our findings indicate that IgG glycan hydrolyzing activity is present in SDSD, and that the activity can be attributed to the here identified enzyme EndoSd.

  8. Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles

    Science.gov (United States)

    Tauber, M. M.; Cavaco-Paulo, A.; Robra, K.-H.; Gübitz, G. M.

    2000-01-01

    Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein−1) and amidase activity (38.4 nkat mg of protein−1) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C.I. Basic Blue 9. PMID:10742253

  9. Liquid Chromatography-Mass Spectrometry Analysis Reveals Hydrolyzed Gluten in Beers Crafted To Remove Gluten.

    Science.gov (United States)

    Colgrave, Michelle L; Byrne, Keren; Howitt, Crispin A

    2017-11-08

    During brewing, gluten proteins may be solubilized, modified, complexed, hydrolyzed, and/or precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with celiac disease (CD) or gluten intolerance. Barley-based beers crafted to remove gluten using proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented products remains controversial. The industry standard, a competitive ELISA, may indicate gluten values gluten peptides derived from hydrolyzed fragments, many >30 kDa in size. Barley gluten (hordeins) were detected in all beers analyzed with peptides representing all hordein classes detected in conventional beers but also, alarmingly, in many gluten-reduced beers. It is evident that PEP digestion was incomplete in several commercial beers, and peptides comprising missed cleavages were identified, warranting further optimization of PEP application in an industrial setting.

  10. High Antioxidant Action and Prebiotic Activity of Hydrolyzed Spent Coffee Grounds (HSCG) in a Simulated Digestion-Fermentation Model: Toward the Development of a Novel Food Supplement.

    Science.gov (United States)

    Panzella, Lucia; Pérez-Burillo, Sergio; Pastoriza, Silvia; Martín, María Ángeles; Cerruti, Pierfrancesco; Goya, Luis; Ramos, Sonia; Rufián-Henares, José Ángel; Napolitano, Alessandra; d'Ischia, Marco

    2017-08-09

    Spent coffee grounds are a byproduct with a large production all over the world. The aim of this study was to explore the effects of a simulated digestion-fermentation treatment on hydrolyzed spent coffee grounds (HSCG) and to investigate the antioxidant properties of the digestion and fermentation products in the human hepatocellular carcinoma HepG2 cell line. The potentially bioaccessible (soluble) fractions exhibited high chemoprotective activity in HepG2 cells against oxidative stress. Structural analysis of both the indigestible (insoluble) and soluble material revealed partial hydrolysis and release of the lignin components in the potentially bioaccessible fraction following simulated digestion-fermentation. A high prebiotic activity as determined from the increase in Lactobacillus spp. and Bifidobacterium spp. and the production of short-chain fatty acids (SCFAs) following microbial fermentation of HSCG was also observed. These results pave the way toward the use of HSCG as a food supplement.

  11. Dark hydrogen fermentation from hydrolyzed starch treated with recombinant amylase originating from Caldimonas taiwanensis On1.

    Science.gov (United States)

    Chen, Shing-Der; Sheu, Der-Shyan; Chen, Wen-Ming; Lo, Yung-Chung; Huang, Tian-I; Lin, Chiu-Yue; Chang, Jo-Shu

    2007-01-01

    Starch is one of the most abundant resources on earth and is suited to serve as a cost-effective feedstock for biological hydrogen production. However, producing hydrogen from direct fermentation of starch is usually inefficient, as the starch hydrolysis is often the rate-limiting step. Therefore, in the present work, enzymatic starch hydrolysis was conducted to enhance the feasibility of using starch feedstock for H2 production. The amylase (with a molecular weight of ca. 112 kDa) used for starch hydrolysis was produced from a recombinant E. coli harboring an amylase gene originating from Caldimonas taiwanensis On1. Using statistical experimental design, the optimal pH and temperature for starch hydrolysis with the recombinant amylase was pH 6.86 and 52.4 degrees C, respectively, at an initial starch concentration of 7 g/L. The hydrolyzed products contained mainly glucose, maltotriose, and maltotetrose, while a tiny amount of maltose was also detected. The enzymatically hydrolyzed products of soluble starch and cassava starch were used as the substrate for dark hydrogen fermentation using Clostridium butyricum CGS2 and Clostridium pasteurianum CH4. The highest H2 production rate (vH2) and yield (YH2) of C. butyricum CGS2 was 124.0 mL/h/L and 6.32 mmol H2/g COD, respectively, both obtained with the hydrolysate of cassava starch. The best H2 production rate (63.0 mL/h/L) of C. pasteurianum CH4 occurred when using hydrolyzed cassava starch as the substrate, whereas the highest yield (9.95 mmol H2/g COD) was obtained with the hydrolyzed soluble starch.

  12. Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells.

    Science.gov (United States)

    Raabe, O; Reich, C; Wenisch, S; Hild, A; Burg-Roderfeld, M; Siebert, H-C; Arnhold, S

    2010-12-01

    Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.

  13. Roles of ATP and NADPH in formation of the Fe-S cluster of spinach ferredoxin. [Spinacia oleracea

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Yasuhiro; Mitsui, Akira; Fujita, Yuichi; Matsubara, Hiroshi (Osaka Univ. (Japan))

    1991-01-01

    The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of ({sup 35}S)sulfide or iron, or in Fe-S cluster formation itself. ({sup 35}S)Sulfide was liberated from ({sup 35}S)cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of ({sup 35}S)cysteine occurred before the formation of the {sup 35}S-labeled Fe-S cluster, and the amount of radioactivity in ({sup 35}S)sulfide was greater than that in {sup 35}S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na{sub 2}S) inhibited competitively formation of the {sup 35}S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of ({sup 35}S)sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.

  14. Enhancement of umami taste of hydrolyzed protein from wheat gluten by β-cyclodextrin.

    Science.gov (United States)

    Wang, Lihua; Xu, Baocai; Li, Linlin; Zhang, Mengke; Feng, Tao; Wang, Jinpeng; Jin, Zhengyu

    2016-10-01

    Wheat gluten was hydrolyzed by Flavourzyme and Neutrase at pH 7.0 and 50 °C for 8 h with β-cyclodextrin (β-CD) employed in the reaction. The hydrolysates were enzyme deactivated, cooled and centrifuged at 1500 × g for 15 min. Sensory and chemical characterization of wheat gluten hydrolysates WGH-1 (reaction conducted without β-CD), WGH-2 (reaction conducted with β-CD) and WGH-3 (β-CD added to WGH-1) was performed. WGH-2 revealed enhanced umami taste and higher hydrolyzing degree, total free amino acid amount, protein yield and umami taste amino acid (Glu + Asp) amount. High-performance liquid chromatography showed that the proportion of molecular weight 180-500 Da in WGH-2 was 11.5% higher than that in WGH-1. Further research indicated that β-CD had multiple effects on the hydrolysis. It could not only increase the solubility of wheat gluten but also form inclusion complexes with resultants. This can both promote the hydrolysis and protect oligopeptides from degradation. β-CD was found to have the ability to increase the umami taste of enzyme-hydrolyzed vegetable protein from wheat gluten. The reasons analyzed were that β-CD could take part in the hydrolysis process by improving the solubility of wheat gluten and form inclusion complexes with resultants. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  15. The Prospect of Hydrolyzed Feather Meal as Ruminant Feeds Through Protein Quality Improvement by Microbes

    Directory of Open Access Journals (Sweden)

    CH Prayitno

    2003-01-01

    Full Text Available The waste of the broiler processing (feather is a potential source for animal feed. However the presence of keratins cause limited of feather use. Before using, therefore, feather must be treated to hydrolyze cysteine disulfide bound dominating keratins protein. Enzymatic (biological treatment using microbes will produce specific feather hydrolyzed and does not have negative impact on environment. The research objected to get the microbes which degradated selected keratins, improve protein quality of feather meal and find out the best ration formulation true in vitro the basic information to formulate in vivo ration. The research has been done in Laboratory of Animal Feedstuff Faculty of Animal Science UNSOED for eight months. Fermentation trial was done on liquid media with bath system. In vitro trial used of Tilley and Terry methods with parameter observe was dry matter digestibility, organic matter digestibility, protein degradation, total VFA and solubility in pepsin. Based on all parameter, on fermentation trial with Bacillus licheniformis decides broiler chicken feather had good prospect to be developed on feed protein source. In vitro trial recommended ration with formulation of fermented feather meal concentrate (15 percent, soybeans meal (5 percent, rice bran (20 percent, molasses (4 percent, mineral mix (1 percent, with forage: concentrate ratio 40 : 60 could be used as in vivo ration. (Animal Production 5(1: 19-24 (2003   Key words : Hydrolyze, Feather, Keratin, Digestibility, Ruminant

  16. Protective Effect of Encapsulation in Fermentation of Limonene-contained Media and Orange Peel Hydrolyzate

    Directory of Open Access Journals (Sweden)

    Mohammad J. Taherzadeh

    2007-08-01

    Full Text Available This work deals with the application of encapsulation technology to eliminateinhibition by D-limonene in fermentation of orange wastes to ethanol. Orange peel wasenzymatically hydrolyzed with cellulase and pectinase. However, fermentation of thereleased sugars in this hydrolyzate by freely suspended S. cerevisiae failed due to inhibitionby limonene. On the other hand, encapsulation of S. cerevisiae in alginate membranes wasa powerful tool to overcome the negative effects of limonene. The encapsulated cells wereable to ferment the orange peel hydrolyzate in 7 h, and produce ethanol with a yield of 0.44g/g fermentable sugars. Cultivation of the encapsulated yeast in defined medium wassuccessful, even in the presence of 1.5% (v/v limonene. The capsules’ membranes wereselectively permeable to the sugars and the other nutrients, but not limonene. While1% (v/v limonene was present in the culture, its concentration inside the capsules was notmore than 0.054% (v/v.

  17. Understanding the Role of Physical Properties of Cellulose on Its Hydrolyzability by Cellulases

    Science.gov (United States)

    O'Dell, Patrick Jonathan

    Cellulose has long been explored as a potential feedstock for biofuel, however the recalcitrance of cellulose makes its conversion into biofuel much more challenging and economically unfavorable compared to well-established processes for converting starch or sugar feedstocks into biofuel. Enzymes capable of hydrolyzing cellulose into soluble sugars, glucose and cellobiose, have been found to work processively along cellulose microfibrils starting from reducing end groups. For this study, cellulose was produced and purified in-house from Gluconacetobacter xylinum cultures, and characterized by quantifying functional groups (aldehyde, ketone, and carboxyl groups) to determine the extent of oxidation of cellulose due to the processing steps. The main goal of this study was to look at the impacts of ultrasonication on cellulose's structure and the enzymatic hydrolyzability of cellulose. A completely randomized experimental design was used to test the effect of ultrasonication time and amplitude (intensity) on changes in cellulose fibril length, degree of polymerization, and rates and extents of hydrolysis. Results indicated that sonication time does significantly impact both the fibril length and average degree of polymerization of cellulose. The impact of ultrasonication on the hydrolyzability of cellulose by commercial cellulase and beta-glucosidase preparations could not be effectively resolved due to high variability in the experimental results. These studies serve as a basis for future studies understanding the role of cellulose microstructure in the mechanism of cellulase hydrolysis of cellulose.

  18. Research observation: Hydrolyzable and condensed tannins in plants of the northwest

    Science.gov (United States)

    Gonzalez-Hernandez, M. P.; Karchesy, J.; Starkey, Edward E.

    2003-01-01

    Tannins are secondary metabolites that may influence feeding by mammals on plants. We analyzed hydrolyzable and condensed tannins in 30 plant species consumed by livestock and deer, as a preliminary attempt to study their possible implications on browsing and grazing in forest ecosystems. Heathers (Ericaceae) and plants of the Rose (Rosaceae) family had tannins, while forbs, grasses and shrubs other than the heathers did not show astringency properties. We found the highest tannin content of all the species in Rubus sp., with the highest value around 180 mg TAE/g dry weight in spring. Potentilla erecta, Alnus glutinosa and Quercus robur were next with 57 to 44 mg TAE/g dw. Total tannins in heathers ranged from 22 to 36 mg TAE/g dw. Levels of condensed tannins were higher than hydrolyzable for most of the species. Only Betula alba, Calluna vulgaris, Pteridium aquilinum and Vaccinium myrtillus had 100% hydrolyzable tannins. Tannin content of the species changed seasonally with highest values during the growing season, corresponding to late winter or early spring, depending on the species.

  19. Research observation: Hydrolyzable and condensed tannins in plants of northwest Spain forests

    Science.gov (United States)

    Gonzalez-Hernandez, M. P.; Karchesy, J.; Starkey, E.E.

    2003-01-01

    Tannins are secondary metabolites that may influence feeding by mammals on plants. We analyzed hydrolyzable and condensed tannins in 30 plant species consumed by livestock and deer, as a preliminary attempt to study their possible implications on browsing and grazing in forest ecosystems. Heathers (Ericaceae) and plants of the Rose (Rosaceae) family had tannins, while forbs, grasses and shrubs other than the heathers did not show astringency properties. We found the highest tannin content of all the species in Rubus sp., with the highest value around 180 mg TAE/g dry weight in spring. Potentilla erecta, Alnus glutinosa and Quercus robur were next with 57 to 44 mg TAE/g dw. Total tannins in heathers ranged from 22 to 36 mg TAE/g dw. Levels of condensed tannins were higher than hydrolyzable for most of the species. Only Betula alba, Calluna vulgaris, Pteridium aquilinum and Vaccinium myrtillus had 100% hydrolyzable tannins. Tannin content of the species changed seasonally with highest values during the growing season, corresponding to late winter or early spring, depending on the species.

  20. Efficient dark fermentative hydrogen production from enzyme hydrolyzed rice straw by Clostridium pasteurianum (MTCC116).

    Science.gov (United States)

    Srivastava, Neha; Srivastava, Manish; Kushwaha, Deepika; Gupta, Vijai Kumar; Manikanta, Ambepu; Ramteke, P W; Mishra, P K

    2017-08-01

    In the present work, production of hydrogen via dark fermentation has been carried out using the hydrolyzed rice straw and Clostridium pasteurianum (MTCC116). The hydrolysis reaction of 1.0% alkali pretreated rice straw was performed at 70°C and 10% substrate loading via Fe3O4/Alginate nanocomposite (Fe3O4/Alginate NCs) treated thermostable crude cellulase enzyme following the previously established method. It is noticed that under the optimized conditions, at 70°C the Fe3O4/Alginate NCs treated cellulase has produced around 54.18g/L sugars as the rice straw hydrolyzate. Moreover, the efficiency of the process illustrates that using this hydrolyzate, Clostridium pasteurianum (MTCC116) could produce cumulative hydrogen of 2580ml/L in 144h with the maximum production rate of 23.96ml/L/h in 96h. In addition, maximum dry bacterial biomass of 1.02g/L and 1.51g/L was recorded after 96h and 144h, respectively with corresponding initial pH of 6.6 and 3.8, suggesting higher hydrogen production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Digestion of feed fractions and intake of heifers fed hydrolyzed sugarcane stored for different periods

    Directory of Open Access Journals (Sweden)

    Regis Luis Missio

    2012-07-01

    Full Text Available The objective of this study was to evaluate, in Nellore heifers, intake and digestibility of hydrolyzed sugarcane stored for different periods. The experimental design used was a 4 × 4 Latin square, four diets, four Nellore heifers with ruminal cannulas (initial body weight 285.4±23.08 kg and average initial age 14 months and four periods of 21 days. The diets were composed by fresh sugarcane (time zero or hydrolyzed sugarcane with addition of 0.5% of hydrated lime, stored for 24, 48 or 72 hours, as the unique forage. Intake and digestibility of feed fractions, nitrogen balance, microbial synthesis efficiency, total number of ruminal protozoans and ammoniacal nitrogen did not significantly change by storing sugarcane with addition of 0.5% of hydrated lime. Sugarcane pH varied quadratically for storage time, with maximum pH of 7.02 after 24 hours from lime addition. Ruminal liquid pH values were higher for heifers fed fresh sugarcane, in comparison with those fed hydrolyzed sugarcane. Sugarcane treated with 0.5% of hydrated lime stored for up to 72 hours does not change ruminal digestion to alter the amount of feed consumed by pubescent Nellore heifers. Thus, lime is a viable technology, once it allows long-duration storage and bee control on treated forage, which contributes to animal feeding logistics.

  2. Improvement of Ethanol Fermentation of Corn Semolina Hydrolyzates with Immobilized Yeast by Medium Supplementation

    Directory of Open Access Journals (Sweden)

    Svetlana Nikolić

    2009-01-01

    Full Text Available The possibilities of improving ethanol fermentation of enzymatically obtained corn semolina hydrolyzates with alginate-immobilized yeast Saccharomyces cerevisiae var. ellipsoideus by medium supplementation with mineral salts as sources of magnesium, zinc, calcium and copper ions, and vitamins (pantothenate, thiamine, pyridoxine, biotin and inositol, separately or as combined mixtures, have been investigated. Among all tested minerals, alone or combined, the most efficient in improving ethanol productivity during fermentation of corn semolina hydrolyzates was a mixture of magnesium and zinc salts: MgSO4 (2 g/L and ZnSO4 (0.3 g/L. Positive effects were also obtained with the addition of copper ions (CuCl2, 1 mg/L or calcium ions (CaCl2, 40 mg/L. Among vitamins, the most effective was Ca-pantothenate (1 g/L, which caused an increase in the fermentation efficiency for approx. 8 %, compared to the control sample. Based on these results, an effective mixture of vitamins and minerals consisting of MgSO4 (2 g/L, ZnSO4 (0.3 g/L, CuCl2 (1 mg/L, Ca-pantothenate (1 g/L and inositol (1 g/L was arranged for the supplementation of the medium based on corn semolina hydrolyzates. The supplementation with this mixture provided an increase of the fermentation efficiency for 20 % compared to the control sample, without supplementation.

  3. Caseinate-gelatin and caseinate-hydrolyzed gelatin composites formed via transglutaminase: chemical and functional properties.

    Science.gov (United States)

    Luo, Zhen-Ling; Zhao, Xin-Huai

    2015-11-01

    Treatment of food proteins by enzymatic crosslinking and other reactions can confer modified properties on the treated proteins. Bovine gelatin and hydrolyzed bovine gelatin were used to generate two caseinate-based composites via transglutaminase, and potential useful properties to food processing were investigated for both composites. Caseinate-gelatin and caseinate-hydrolyzed gelatin composites contained 33.4 and 10.3 g kg(-1) protein of 4-hydroxyproline, respectively. Caseinate conjugation with gelatin and hydrolyzed gelatin resulted in two composites with stronger absorption at five wavenumbers during Fourier transform-infrared analysis, demonstrating that they were rich in hydroxyl and carboxyl groups. Both composites exhibited higher viscosity values in aqueous dispersions, lower thermal stability (i.e. higher mass loss) during thermogravimetric analysis and worse emulsifying properties than original caseinate, owing to conjugation and crosslinking via transglutaminase. However, confocal laser scanning microscopy (CLSM) analysis revealed that both composites actually had better emulsion stability after 2 weeks of storage. The composites generated were different in chemical characteristics and better in viscosity and emulsion stability than original caseinate. They might have potential as protein thickeners and emulsifiers. CLSM is a better technique to assess emulsion stability of food proteins than the classic turbidity method. © 2014 Society of Chemical Industry.

  4. Tolerance of a standard intact protein formula versus a partially hydrolyzed formula in healthy, term infants

    Directory of Open Access Journals (Sweden)

    Marunycz John D

    2009-06-01

    Full Text Available Abstract Background Parents who perceive common infant behaviors as formula intolerance-related often switch formulas without consulting a health professional. Up to one-half of formula-fed infants experience a formula change during the first six months of life. Methods The objective of this study was to assess discontinuance due to study physician-assessed formula intolerance in healthy, term infants. Infants (335 were randomized to receive either a standard intact cow milk protein formula (INTACT or a partially hydrolyzed cow milk protein formula (PH in a 60 day non-inferiority trial. Discontinuance due to study physician-assessed formula intolerance was the primary outcome. Secondary outcomes included number of infants who discontinued for any reason, including parent-assessed. Results Formula intolerance between groups (INTACT, 12.3% vs. PH, 13.7% was similar for infants who completed the study or discontinued due to study physician-assessed formula intolerance. Overall study discontinuance based on parent- vs. study physician-assessed intolerance for all infants (14.4 vs.11.1% was significantly different (P = 0.001. Conclusion This study demonstrated no difference in infant tolerance of intact vs. partially hydrolyzed cow milk protein formulas for healthy, term infants over a 60-day feeding trial, suggesting nonstandard partially hydrolyzed formulas are not necessary as a first-choice for healthy infants. Parents frequently perceived infant behavior as formula intolerance, paralleling previous reports of unnecessary formula changes. Trial Registration clinicaltrials.gov: NCT00666120

  5. Breast feeding increases vasoconstriction induced by electrical field stimulation in rat mesenteric artery. Role of neuronal nitric oxide and ATP.

    Directory of Open Access Journals (Sweden)

    Javier Blanco-Rivero

    Full Text Available OBJECTIVES: The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF affects the vasomotor response induced by electrical field stimulation (EFS, participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications. METHODS: Experiments were performed in female Sprague-Dawley rats (3 months old, divided into three groups: Control (in oestrous phase, mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase. Vasomotor response to EFS, noradrenaline (NA and nitric oxide (NO donor DEA-NO were studied. Neuronal NO synthase (nNOS and phosphorylated nNOS (P-nNOS protein expression were analysed and NO, superoxide anion (O(2(.-, NA and ATP releases were also determined. RESULTS: EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O(2(.- production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals. CONCLUSION: Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.

  6. Hindering the strand passage reaction of human topoisomerase IIalpha without disturbing DNA cleavage, ATP hydrolysis, or the operation of the N-terminal clamp

    DEFF Research Database (Denmark)

    Oestergaard, Vibe H; Giangiacomo, Laura; Bjergbaek, Lotte

    2004-01-01

    DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent...

  7. 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

    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...... 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...... connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U...

  8. Extensively Hydrolyzed Formula (MA-mi Induced Exacerbation of Food Protein-Induced Enterocolitis Syndrome (FPIES in a Male Infant

    Directory of Open Access Journals (Sweden)

    Tomoyuki Kabuki

    2007-01-01

    Discussion: MA-mi is likely to be used increasingly for allergic infants, but it is not necessarily a substitute for other hydrolyzed milk formulae in all cases, and care should be taken regarding its use and possible misuse.

  9. Eggshell membrane hydrolyzates activate NF-κB in vitro: possible implications for in vivo efficacy

    Directory of Open Access Journals (Sweden)

    Ruff KJ

    2015-02-01

    Full Text Available Kevin J Ruff,1 Paul L Durham,2 Austin O’Reilly,2 F Daniel Long1 1ESM Technologies, LLC, Carthage, MO, USA; 2Center for Biomedical and Life Sciences, Missouri State University, Springfield, MO, USA Purpose: Eggshell membrane (ESM has been shown to contain naturally occurring bioactive components, and biological activities such as reducing proinflammatory cytokines, liver fibrosis, and joint pain in osteoarthritis sufferers have also been reported for ESM matrix as a whole. Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB is a signaling protein found in the cytoplasm of nearly all human and animal cell types and is a primary regulator of immune function. The studies reported herein were designed to investigate the possible role that NF-κB activity might play in the reported biological activities of ESM. Methods: Three ESM hydrolyzates produced via fermentation, enzymatic, or chemical hydrolysis were evaluated in vitro in either human peripheral blood mononuclear cell or THP-1 (human leukemic monocyte cell cultures for NF-κB activity following 4-hour exposure. The hydrolyzates were compared with untreated control cells or cells incubated with lipopolysaccharide or ascorbic acid. The source of ESM activity was also evaluated. Results: NF-κB levels were increased above levels found in untreated cells at all three dilutions (1:100, 1:1,000, and 1:10,000 for the fermentation hydrolyzate of ESM (ESM-FH (P=0.021, P=0.020, P=0.009, respectively in peripheral blood mononuclear cells. The enzymatic hydrolyzate of ESM (ESM-EH also produced statistically significant levels of activated NF-κB at the 1:100 and 1:1,000 dilutions (P=0.004, P=0.006, respectively but fell just shy of significance at the 1:10,000 dilution (P=0.073. Similarly, ESM-FH (P=0.021, P=0.002 and ESM-EH (P=0.007, P=0.007 activated NF-κB in THP-1 cells at 1:1,000 and 1:10,000 dilutions, respectively. The chemical hydrolyzate of ESM (ESM-CH showed statistically

  10. Distinct neurological disorders with ATP1A3 mutations

    Science.gov (United States)

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

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na+/K+-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in vitro and animal model systems, and the role of Na+/K+-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases. PMID:24739246

  11. A Randomized Controlled Trial Assessing Growth of Infants Fed a 100% Whey Extensively Hydrolyzed Formula Compared With a Casein-Based Extensively Hydrolyzed Formula

    Directory of Open Access Journals (Sweden)

    David Fields PhD

    2016-04-01

    Full Text Available This study compared the growth of healthy infants fed a hypoallergenic 100% whey-based extensively hydrolyzed formula (EHF with Bifidobacterium lactis (test with that of infants fed an extensively hydrolyzed casein formula (control. Formula-fed infants (14 ± 3 days were randomized to test or control groups until 112 days of age. Anthropometrics were assessed at 14, 28, 56, 84, and 112 days, and daily records were kept for 2 days prior to study visits. Serum albumin and plasma amino acids at 84 days were assessed in a subset. A total of 282 infants were randomized (124 test, 158 control. Significantly more infants dropped out of the control (56% as compared with the test (41% group. Mean daily weight gain was significantly higher in the test group compared with the control group (27.95 ± 5.91 vs 25.93 ± 6.12 g/d; P = .027 with the test group reporting significantly fewer stools (2.2 vs 3.6 stools/d; P 3 loose stools/d and a higher incidence of vomiting as compared with the test group. There were no differences in gas, mood, sleep, or serum albumin. Plasma arginine and valine were significantly lower in the test group, whereas leucine and lysine were higher; all values were within normal limits. Significantly more adverse events attributed to the study formula were reported in the control group. The 100% whey-based hypoallergenic EHF containing Bifidobacterium lactis and medium chain triglycerides supported growth of healthy infants. Future studies on the application of this formula in clinically indicated populations are warranted.

  12. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. FIBROBLAST CYTOSKELETAL REMODELING INDUCED BY TISSUE STRETCH INVOLVES ATP SIGNALING

    OpenAIRE

    Langevin, HM; Fujita, T.; Bouffard, NA; Takano, T; Koptiuch, C; Badger, GJ; Nedergaard, M

    2013-01-01

    Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 minutes, returning to baseline at 60 min...

  14. YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes.

    Science.gov (United States)

    Handa, Yoshihiro; Inaho, Noriyuki; Nameki, Nobukazu

    2011-03-01

    In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

  15. Sensory aroma characteristics of alcalase hydrolyzed rice bran protein concentrate as affected by spray drying and sugar addition.

    Science.gov (United States)

    Arsa, Supeeraya; Theerakulkait, Chockchai

    2015-08-01

    The sensory aroma characteristics of alcalase hydrolyzed rice bran protein concentrate as affected by spray drying and sugar addition were investigated. Rice bran protein concentrate (RBPC) was hydrolyzed by alcalase. Sucrose, glucose or fructose was added to the liquid rice bran protein hydrolysate (LRBPH) and subsequently spray dried. The sensory aroma intensities of the hydrolysates were evaluated. Results showed that after spray drying, the rice bran protein concentrate powder (RBPC-P) had higher sweet and cocoa-like aroma intensities than RBPC (p ≤ 0.05) and hydrolyzed rice bran protein powder (HRBPP) had higher milk powder-like aroma intensities than LRBPH (p ≤ 0.05). The sweet, cocoa-like and milk powder-like aroma intensities in hydrolyzed rice bran protein powder with fructose addition (HRBPP-F) were significantly higher (p ≤ 0.05) than those of hydrolyzed rice bran protein powder with sucrose or glucose addition (HRBPP-S or HRBPP-G). HRBPP-F had the highest overall aroma liking score. These results also indicate that spray drying and sugar addition could improve the sensory aroma characteristics of alcalase hydrolyzed RBPC.

  16. Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol.

    Science.gov (United States)

    Nilsson, A; Taherzadeh, M J; Lidén, G

    2001-07-26

    Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(E,max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations.

  17. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  18. F1F0 ATP Synthase–Cyclophilin D Interaction Contributes to Diabetes-Induced Synaptic Dysfunction and Cognitive Decline

    Science.gov (United States)

    Yan, Shijun; Du, Fang; Wu, Long; Zhang, Zhihua; Zhong, Changjia; Yu, Qing; Wang, Yongfu; Lue, Lih-Fen; Walker, Douglas G.; Douglas, Justin T.

    2016-01-01

    Mitochondrial abnormalities are well known to cause cognitive decline. However, the underlying molecular basis of mitochondria-associated neuronal and synaptic dysfunction in the diabetic brain remains unclear. Here, using a mitochondrial single-channel patch clamp and cyclophilin D (CypD)-deficient mice (Ppif −/−) with streptozotocin-induced diabetes, we observed an increase in the probability of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in brain mitochondria of diabetic mice, which was further confirmed by mitochondrial swelling and cytochrome c release induced by Ca2+ overload. Diabetes-induced elevation of CypD triggers enhancement of F1F0 ATP synthase–CypD interaction, which in turn leads to mPTP opening. Indeed, in patients with diabetes, brain cypD protein levels were increased. Notably, blockade of the F1F0 ATP synthase–CypD interaction by CypD ablation protected against diabetes-induced mPTP opening, ATP synthesis deficits, oxidative stress, and mitochondria dysfunction. Furthermore, the absence of CypD alleviated deficits in synaptic plasticity, learning, and memory in diabetic mice. Thus, blockade of ATP synthase interaction with CypD provides a promising new target for therapeutic intervention in diabetic encephalopathy. PMID:27554467

  19. Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats?

    Science.gov (United States)

    Camara, Henrique; Rodrigues, Juliano Quintella Dantas; Alves, Gabriel Andrade; da Silva Junior, Edilson Dantas; Caricati-Neto, Afonso; Garcia, Antônio G; Jurkiewicz, Aron

    2015-12-05

    Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration-effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000μM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1-300µM and cardiac arrest at 1000μM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. ATP4a is required for development and function of the Xenopus mucociliary epidermis - a potential model to study proton pump inhibitor-associated pneumonia.

    Science.gov (United States)

    Walentek, Peter; Beyer, Tina; Hagenlocher, Cathrin; Müller, Christina; Feistel, Kerstin; Schweickert, Axel; Harland, Richard M; Blum, Martin

    2015-12-15

    Proton pump inhibitors (PPIs), which target gastric H(+)/K(+)ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/β-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Biophysical comparison of ATP-driven proton pumping mechanisms suggests a kinetic advantage for the rotary process depending on coupling ratio.

    Science.gov (United States)

    Anandakrishnan, Ramu; Zuckerman, Daniel M

    2017-01-01

    ATP-driven proton pumps, which are critical to the operation of a cell, maintain cytosolic and organellar pH levels within a narrow functional range. These pumps employ two very different mechanisms: an elaborate rotary mechanism used by V-ATPase H+ pumps, and a simpler alternating access mechanism used by P-ATPase H+ pumps. Why are two different mechanisms used to perform the same function? Systematic analysis, without parameter fitting, of kinetic models of the rotary, alternating access and other possible mechanisms suggest that, when the ratio of protons transported per ATP hydrolyzed exceeds one, the one-at-a-time proton transport by the rotary mechanism is faster than other possible mechanisms across a wide range of driving conditions. When the ratio is one, there is no intrinsic difference in the free energy landscape between mechanisms, and therefore all mechanisms can exhibit the same kinetic performance. To our knowledge all known rotary pumps have an H+:ATP ratio greater than one, and all known alternating access ATP-driven proton pumps have a ratio of one. Our analysis suggests a possible explanation for this apparent relationship between coupling ratio and mechanism. When the conditions under which the pump must operate permit a coupling ratio greater than one, the rotary mechanism may have been selected for its kinetic advantage. On the other hand, when conditions require a coupling ratio of one or less, the alternating access mechanism may have been selected for other possible advantages resulting from its structural and functional simplicity.

  2. 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...... 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...

  3. Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

    Science.gov (United States)

    Nakanishi, Simone C; Soares, Lauren B; Biazi, Luiz Eduardo; Nascimento, Viviane M; Costa, Aline C; Rocha, George Jackson M; Ienczak, Jaciane L

    2017-10-01

    Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L -1  · h -1 (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L -1  · h -1 for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. EndoE from Enterococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth.

    Directory of Open Access Journals (Sweden)

    Julia Garbe

    Full Text Available Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG. In this report we identified the human glycoprotein lactoferrin (hLF as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.

  5. EndoE from Enterococcus faecalis Hydrolyzes the Glycans of the Biofilm Inhibiting Protein Lactoferrin and Mediates Growth

    Science.gov (United States)

    Garbe, Julia; Sjögren, Jonathan; Cosgrave, Eoin F. J.; Struwe, Weston B.; Bober, Marta; Olin, Anders I.; Rudd, Pauline M.; Collin, Mattias

    2014-01-01

    Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism. PMID:24608122

  6. Renin release

    DEFF Research Database (Denmark)

    Schweda, Frank; Friis, Ulla; Wagner, Charlotte

    2007-01-01

    The aspartyl-protease renin is the key regulator of the renin-angiotensin-aldosterone system, which is critically involved in salt, volume, and blood pressure homeostasis of the body. Renin is mainly produced and released into circulation by the so-called juxtaglomerular epithelioid cells, located...

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

    Science.gov (United States)

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

    2008-04-01

    Reconstruction after multilevel decompression of the cervical spine, especially in the weakened osteoporotic, neoplastic or infectious spine often requires circumferential stabilization and fusion. To avoid the additional posterior surgery in these cases while increasing rigidity of anterior-only screw-plate constructs, the authors introduce the concept of anterior transpedicular screw (ATPS) fixation. We demonstrated its morphological feasibility as well as its indications in a previous study in Part I of our project. Consequently, the objectives of the current study were to assess the ex vivo accuracy of placing ATPS into the cervical vertebra as well as the biomechanical performance of ATPS in comparison to traditional vertebral body screws (VBS) in terms of pull-out strength (POS). Twenty-three ATPS were inserted alternately to two screws into the pedicles and vertebral bodies, respectively, of six cadaveric specimens from C3-T1. For insertion of ATPS, a manual fluoroscopically assisted technique was used. Pre- and post insertional CT-scans were used to assess accuracy of ATPS insertion in the axial and sagittal planes. A newly designed grading system and accuracy score were used to delineate accuracy of ATPS insertion. Following insertion of screws, 23 ATPS and 22 VBS were subjected to pull-out testing (POT). The bone mineral density (BMD) of each specimen was assessed prior to POT. Statistical analysis showed that the incidence of correctly placed screws and non-critical pedicles breaches in axial plane was 78.3%, and 95.7% in sagittal plane. Hence, according to our definition of "critical" pedicle breach that exposes neurovascular structures at risk, 21.7% (n = 5) of all ATPS inserted showed a critical pedicle breach in axial plane. Notably, no critical pedicle perforation occurred at the C6 to T1 levels. Pull-out testing of ATPS and VBS revealed that pull-out resistance of ATPS was 2.5-fold that of VBS. Mean POS of 23 ATPS with a mean BMD of 0.566 g/cm(2

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

    Science.gov (United States)

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

    2015-01-01

    -helix of γ-subunit that acts as a lift; then, in the formation of phosphoryl group; and lastly, in the release of ATP molecules from the active center of the enzyme and the loading of ADP. We are aware that our model is not an accepted model for ATP synthesis, but it is presented here for further examination and test.

  9. Dietary Plant Sterol Esters Must Be Hydrolyzed to Reduce Intestinal Cholesterol Absorption in Hamsters.

    Science.gov (United States)

    Carden, Trevor J; Hang, Jiliang; Dussault, Patrick H; Carr, Timothy P

    2015-07-01

    Elevated concentrations of LDL cholesterol are associated with the development of atherosclerosis and therefore are considered an important target for intervention to prevent cardiovascular diseases. The inhibition of cholesterol absorption in the small intestine is an attractive approach to lowering plasma cholesterol, one that is addressed by drug therapy as well as dietary supplementation with plant sterols and plant sterol esters (PSEs). This study was conducted to test the hypothesis that the cholesterol-lowering effects of PSE require hydrolysis to free sterols (FSs). Male Syrian hamsters were fed atherogenic diets (AIN-93M purified diet containing 0.12% cholesterol and 8% coconut oil) to which one of the following was added: no PSEs or ethers (control), 5% sterol stearate esters, 5% sterol palmitate esters (PEs), 5% sterol oleate esters (OEs), 5% sterol stearate ethers (STs; to mimic nonhydrolyzable PSE), or 3% FSs plus 2% sunflower oil. The treatments effectively created a spectrum of PSE hydrolysis across which cholesterol metabolism could be compared. Metabolic measurements included cholesterol absorption, plasma and liver lipid concentration, and fecal neutral sterol and bile acid excretion. The STs and the PEs and SEs were poorly hydrolyzed (1.69-4.12%). In contrast, OEs were 88.3% hydrolyzed. The percent hydrolysis was negatively correlated with cholesterol absorption (r = -0.85; P cholesterol excretion (r = 0.92; P cholesterol-lowering properties of PSE. Our data on hamsters suggest that PSE hydrolysis and the presence of FSs is necessary to induce an optimum cholesterol-lowering effect and that poorly hydrolyzed PSEs may lower cholesterol through an alternative mechanism than that of competition with cholesterol for micelle incorporation. © 2015 American Society for Nutrition.

  10. Dietary Plant Sterol Esters Must Be Hydrolyzed to Reduce Intestinal Cholesterol Absorption in Hamsters123

    Science.gov (United States)

    Carden, Trevor J; Hang, Jiliang; Dussault, Patrick H; Carr, Timothy P

    2015-01-01

    Background: Elevated concentrations of LDL cholesterol are associated with the development of atherosclerosis and therefore are considered an important target for intervention to prevent cardiovascular diseases. The inhibition of cholesterol absorption in the small intestine is an attractive approach to lowering plasma cholesterol, one that is addressed by drug therapy as well as dietary supplementation with plant sterols and plant sterol esters (PSEs). Objective: This study was conducted to test the hypothesis that the cholesterol-lowering effects of PSE require hydrolysis to free sterols (FSs). Methods: Male Syrian hamsters were fed atherogenic diets (AIN-93M purified diet containing 0.12% cholesterol and 8% coconut oil) to which one of the following was added: no PSEs or ethers (control), 5% sterol stearate esters, 5% sterol palmitate esters (PEs), 5% sterol oleate esters (OEs), 5% sterol stearate ethers (STs; to mimic nonhydrolyzable PSE), or 3% FSs plus 2% sunflower oil. The treatments effectively created a spectrum of PSE hydrolysis across which cholesterol metabolism could be compared. Metabolic measurements included cholesterol absorption, plasma and liver lipid concentration, and fecal neutral sterol and bile acid excretion. Results: The STs and the PEs and SEs were poorly hydrolyzed (1.69–4.12%). In contrast, OEs were 88.3% hydrolyzed. The percent hydrolysis was negatively correlated with cholesterol absorption (r = −0.85; P cholesterol excretion (r = 0.92; P cholesterol-lowering properties of PSE. Conclusions: Our data on hamsters suggest that PSE hydrolysis and the presence of FSs is necessary to induce an optimum cholesterol-lowering effect and that poorly hydrolyzed PSEs may lower cholesterol through an alternative mechanism than that of competition with cholesterol for micelle incorporation. PMID:25972524

  11. Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides

    Science.gov (United States)

    Zhang, Jun; Yin, Jin-Gang; Hang, Bao-Jian; Cai, Shu; Li, Shun-Peng

    2012-01-01

    The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with Km and kcat values of 29.5 μM and 49.2 s−1, respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments. PMID:22544249

  12. ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes

    NARCIS (Netherlands)

    de Waart, Dirk R.; Naik, Jyoti; Utsunomiya, Karina S.; Duijst, Suzanne; Ho-Mok, Kam; Bolier, A. Ruth; Hiralall, Johan; Bull, Laura N.; Bosma, Piter J.; Oude Elferink, Ronald P. J.; Paulusma, Coen C.

    2016-01-01

    ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a

  13. Rheological properties of disperse systems based on hydrolyzed lignin and oil

    Science.gov (United States)

    Savitskaya, T. A.; Reznikov, I. V.; Shcheglov, V. A.; Tsygankova, N. G.; Telysheva, G. M.; Grinshpan, D. D.

    2012-05-01

    The rheological properties of oil dispersions of hydrophobized hydrolyzed lignin, which is an effective sorbent of oil and oil products, have been studied. It has been shown that at a lignin concentration of over 10%, free-disperse systems with Newton flow go over to coagulation thixotropic structures, demonstrating the pseudoplastic character of flow. The structural-mechanical characteristics of highly filled (lignin content of 40-45%) dispersions have been determined and the concentration region of existence of an oil-saturated sorbent in the form of a solid product easily removed from the water surface has been established.

  14. FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

    Science.gov (United States)

    Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

    2013-07-01

    Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

  15. Enhancing ethanol fermentability of an artificial acid hydrolyzate with anion exchange resin treatment.

    Science.gov (United States)

    Zhang, Yi; Gao, Jun; Ntoni, Jennifer; Begonia, Maria F T; Lee, Ken S; Hwang, Huey-Min

    2008-01-01

    To assess the effectiveness of anion exchange resins (Dowex M43 and Dowex monosphere 66) in neutralization and detoxification of an acid hydrolyzate solution, a fermentation medium containing inhibitors was inoculated with Saccharomyces cerevisiae. When treated with resins at a 1:1 ratio (vol:wt) for up to 20 min, 55-67% of furan and more than 95% of phenolic compounds were removed. Ethanol fermentation activity in resin-treated fermentation medium was the same as the control. There was 21-43% of the total sugar loss after one resin treatment, depending on the sugar concentration. Additional treatments increased sugar retention rate to 95%.

  16. Individual actin filaments in a microfluidic flow reveal the mechanism of ATP hydrolysis and give insight into the properties of profilin.

    Directory of Open Access Journals (Sweden)

    Antoine Jégou

    2011-09-01

    Full Text Available The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.

  17. Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers.

    Science.gov (United States)

    1986-01-01

    currents and Mode 2 displays currents which are long-lasting. Mode 0 can be achieved with blockers and Mode 2 with activators such as Bay K 8644, CGP ...vertebrate red blood e. %, cells. Amer. Zool. 20:103-114, 1980. 24. Bartlett, G.R. Phosphate compounds in red cells of reptiles , amphibians and fish. Comp...Comp. Biochem. Physiol. 43A:975-989, 1972. 194. Hartman, F.A. and Lessler, M.A. Erythrocyte measurements in fishes, amphibia, and reptiles . Biol. Bull

  18. Piperine Suppresses Pyroptosis and Interleukin-1β Release upon ATP Triggering and Bacterial Infection

    OpenAIRE

    Yi-Dan Liang; Wen-Jing Bai; Chen-Guang Li; Li-Hui Xu; Hong-Xia Wei; Hao Pan; Xian-Hui He; Dong-Yun Ouyang

    2016-01-01

    Piperine is a phytochemical present in black pepper (Piper nigrum Linn) and other related herbs, possessing a wide array of pharmacological activities including anti-inflammatory effects. Previously, we demonstrated that piperine has therapeutic effects on bacterial sepsis in mice, but the underlying mechanism has not been fully elucidated. In this study, we aimed to investigate the influences of piperine on pyroptosis in murine macrophages. The results showed that piperine dose-dependently i...

  19. ATP2A3 gene as an important player for resveratrol anticancer activity in breast cancer cells.

    Science.gov (United States)

    Izquierdo-Torres, Eduardo; Rodríguez, Gabriela; Meneses-Morales, Iván; Zarain-Herzberg, Angel

    2017-07-01

    The Ca2+ -ATPases from the Sarco/endoplasmic reticulum (SERCA) are fundamental for maintaining intracellular [Ca2+ ] homeostasis by pumping Ca2+ into the endoplasmic reticulum (ER) of eukaryotic cells. SERCA enzymes are encoded by three different genes (ATP2A1-3), whose expression occurs in a tissue and development stage-specific manner. It has been reported alterations in the expression of SERCA2 and SERCA3 pumps in different types of cancer: oral, lung, colon, stomach, central nervous system, thyroid, breast, and prostate. Resveratrol (RSV), a phytoalexin produced by a wide variety of plants in response to stress situations can modulate cellular processes involved in all stages of carcinogenesis. In this work, we used breast cancer cell lines (MCF-7 and MDA-MB-231) to evaluate mRNA levels of ATP2A2 and ATP2A3 genes in response to RSV treatment. Our results demonstrate that RSV treatment induced the expression of ATP2A3 gene in both cell lines in a time and concentration-dependent manner, while the expression of ATP2A2 gene remained unaffected. The RSV-induced expression of SERCA3 in these breast cancer cell lines produced decreased cell viability, triggered apoptosis and changes in cytosolic Ca2+ levels, as well as changes in the capacity for Ca2+ release by the ER. These data suggest an important participation of SERCA3 genes in RSV-mediated anti-tumor effect in breast cancer cell lines. Nevertheless, further research is needed to elucidate the molecular mechanisms underlying this effect. © 2017 Wiley Periodicals, Inc.

  20. Fibroblast cytoskeletal remodeling induced by tissue stretch involves ATP signaling.

    Science.gov (United States)

    Langevin, Helene M; Fujita, Takumi; Bouffard, Nicole A; Takano, Takahiro; Koptiuch, Cathryn; Badger, Gary J; Nedergaard, Maiken

    2013-09-01

    Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 min, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediate the active change in fibroblast shape that occurs in response to a static increase in tissue length. Copyright © 2013 Wiley Periodicals, Inc.

  1. Phenomenological analysis of ATP dependence of motor protein

    CERN Document Server

    Zhang, Yunxin

    2011-01-01

    In this study, through phenomenological comparison of the velocity-force data of processive motor proteins, including conventional kinesin, cytoplasmic dynein and myosin V, we found that, the ratio between motor velocities of two different ATP concentrations is almost invariant for any substall, superstall or negative external loads. Therefore, the velocity of motor can be well approximated by a Michaelis-Menten like formula $V=\\atp k(F)L/(\\atp +K_M)$, with $L$ the step size, and $k(F)$ the external load $F$ dependent rate of one mechanochemical cycle of motor motion in saturated ATP solution. The difference of Michaelis-Menten constant $K_M$ for substall, superstall and negative external load indicates, the ATP molecule affinity of motor head for these three cases are different, though the expression of $k(F)$ as a function of $F$ might be unchanged for any external load $F$. Verifications of this Michaelis-Menten like formula has also been done by fitting to the recent experimental data.

  2. [ATP content in cryopreserved sperm of Siberian white cranes Grus leucogeranus (Aves: Gruiformes)].

    Science.gov (United States)

    Maksudov, G Iu; Erokhin, A S; Nesterenko, O N; Panchenko, V G

    2002-01-01

    ATP contents were studied in the native and cryoconserved sperm of Siberian white cranes Grus leucogeranus using bioluminescence analysis. The ATP content in freshly obtained spermatozoa was 12.7 nmol/10(8) cells. No ATP was found in the seminal plasma. In the process of freezing-thawing, the ATP concentration in the spermatozoa decreased by 30%. The differences in the dynamics of ATP content during cryoconservation of sperm of white cranes and other birds and mammals are discussed.

  3. Aptamer/Graphene Quantum Dots Nanocomposite Capped Fluorescent Mesoporous Silica Nanoparticles for Intracellular Drug Delivery and Real-Time Monitoring of Drug Release.

    Science.gov (United States)

    Zheng, Fen-Fen; Zhang, Peng-Hui; Xi, Yu; Chen, Jing-Jia; Li, Ling-Ling; Zhu, Jun-Jie

    2015-12-01

    Great challenges in investigating the release of drug in complex cellular microenvironments necessitate the development of stimuli-responsive drug delivery systems with real-time monitoring capability. In this work, a smart drug nanocarrier based on fluorescence resonance energy transfer (FRET) is fabricated by capping graphene quantum dots (GQDs, the acceptor) onto fluorescent mesoporous silica nanoparticles (FMSNs, the donor) via ATP aptamer for real-time monitoring of ATP-triggered drug release. Under extracellular conditions, the fluorescence of FMSNs remains in the "off" state in the low ATP level which is unable to trigger the release of drug. Once specifically recognized and internalized into the target tumor cells by AS1411 aptamer, in the ATP-rich cytoplasm, the conformation switch of the ATP aptamer causes the shedding of the GQDs from the nanocarriers, leading to the release of the loaded drugs and consequently severe cytotoxicity. Simultaneously, the fluorescence of FMSNs turns "on" along with the dissociation of GQDs, which allows real-time monitoring of the release of drug from the pores. Such a drug delivery system features high specificity of dual-target recognition with AS1411 and ATP aptamer as well as high sensitivity of the FRET-based monitoring strategy. Thus, the proposed multifunctional ATP triggered FRET-nanocarriers will find potential applications for versatile drug-release monitoring, efficient drug transport, and targeted cancer therapeutics.

  4. Biological effects of hydrolyzed quinoa extract from seeds of Chenopodium quinoa Willd.

    Science.gov (United States)

    Meneguetti, Quele Adriana; Brenzan, Mislaine Adriana; Batista, Marcia Regina; Bazotte, Roberto Barbosa; Silva, Daniel Rodrigues; Garcia Cortez, Diógenes Aparício

    2011-06-01

    An extract from seeds of Chenopodium quinoa Willd. (quinoa), termed hydrolyzed quinoa (HQ), was obtained by enzymatic hydrolysis from seeds of the quinoa variety BRS-Piabiru. Analysis of the physical and chemical properties of quinoa and HQ showed that the hydrolyzed extract is rich in essential amino acids, particularly those with branched chains (leucine, isoleucine, and valine). In addition, we evaluated the biological effects of HQ, particularly the toxicological potential. For this purpose, male Wistar rats were assigned randomly to four groups: (1) sedentary supplemented group, which received HQ (2,000 mg/kg); (2) sedentary control group, non-supplemented; (3) exercised supplemented group (i.e., rats subjected to aerobic physical exercise that received HQ [2,000 mg/kg]); and (4) exercised control group (i.e., rats subjected to aerobic physical exercise, non-supplemented). After 30 days, all groups were analyzed for levels of serum glucose, cholesterol, triacylglycerol, total protein, albumin, uric acid, and urea and activities of the enzymes alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase. Body weight gain, dietary intake, and lipid deposition were also analyzed. The results showed no hepatic and renal toxicity of HQ. Moreover, decreased food intake, body weight, fat deposition, and blood triacylglycerol level were observed in the supplemented groups (sedentary and exercised supplemented groups). These results suggest a potential use of HQ in human nutrition.

  5. Characterization of Bacterial Mannanase for Hydrolyzing Palm Kernel Cake to Produce Manno-oligosaccharides Prebiotics

    Directory of Open Access Journals (Sweden)

    W. Utami

    2013-12-01

    Full Text Available Palm kernel cake (PKC is a promising source of prebiotics, since it contains high amount of β-mannan which can be further hydrolyzed to manno-oligasaccharides (MOS, a prebiotic. Therefore, this research was carried out to analyze the capability of a bacterial isolate (A2 isolates previously isolated from soils sample from around IPB campus to hydrolyze PKC. Based on 16S-DNA analysis, isolate A2 was identified as Brevibacillus borstelensis. Mannanase of A2 isolate had an optimum condition at 90 oC and pH 7. Mannanase activity of crude extracts using Locust Bean Gum (LBG and PKC as substrates were 0.37U/mL and 0.032U/mL, respectively. However, the most favorable production of oligosaccharides based on the degree of polymerization was obtained after 72-h of incubation with the ratio of substrate:enzyme, 1.2:1, on 1.5% PKC as substrate. The manno-oligosaccharides prebio-tic obtained was found to interfere the growth of both lactic acid bacteria (Lactobacillus casei and pathogenic microflora (Escherichia coli. E. coli apparently could not use this prebiotic as the carbon sources, in contrast to L. casei. Substitution of carbon source in medium with prebiotics reduced the capability of L. casei to produce organic acids. It is concluded that local A2 isolate (B. borstelensis produces mannanase which can be used to produce prebiotics from PKC.

  6. Bioactive Peptides from Angelica sinensis Protein Hydrolyzate Delay Senescence in Caenorhabditis elegans through Antioxidant Activities

    Directory of Open Access Journals (Sweden)

    Qiangqiang Wang

    2016-01-01

    Full Text Available Since excessive reactive oxygen species (ROS is known to be associated with aging and age-related diseases, strategies modulating ROS level and antioxidant defense systems may contribute to the delay of senescence. Here we show that the protein hydrolyzate from Angelica sinensis was capable of increasing oxidative survival of the model animal Caenorhabditis elegans intoxicated by paraquat. The hydrolyzate was then fractionated by ultrafiltration, and the antioxidant fraction (<3 kDa was purified by gel filtration to obtain the antioxidant A. sinensis peptides (AsiPeps, which were mostly composed of peptides with <20 amino acid residues. Further studies demonstrate that AsiPeps were able to reduce the endogenous ROS level, increase the activities of the antioxidant enzymes superoxide dismutase and catalase, and decrease the content of the lipid peroxidation product malondialdehyde in nematodes treated with paraquat or undergoing senescence. AsiPeps were also shown to reduce age pigments accumulation and extend lifespan but did not affect the food-intake behavior of the nematodes. Taken together, our results demonstrate that A. sinensis peptides (AsiPeps are able to delay aging process in C. elegans through antioxidant activities independent of dietary restriction.

  7. Insight into the solvent, temperature and time effects on the hydrogenolysis of hydrolyzed lignin.

    Science.gov (United States)

    Shu, Riyang; Zhang, Qi; Ma, Longlong; Xu, Ying; Chen, Pengru; Wang, Chenguang; Wang, Tiejun

    2016-12-01

    The aim of this study is to explore the reaction mediums and conditions for producing high yield of valuable monomers from concentrated sulfuric acid hydrolyzed lignin. The solvent, temperature and time effects on the hydrogenolysis of hydrolyzed lignin were investigated under the catalysis of Pd/C and CrCl3. Supercritical methanol exhibits the best depolymerization performance, because of its unique diffusion, dissolution and acid-base properties. Afterwards, the influence of reaction temperature and time on depolymerization, repolymerization and coking during hydrogenolysis was examined in methanol. The high temperature is found to favor the depolymerization, with the β-O-4 linkages cleaved significantly. However, the repolymerization is promoted simultaneously, and a high amount of β-β groups form. These reactions are in constant competition with each other and the repolymerization is preferred at excessive high temperature, producing bulk char residues, that is coking. This study will provide a beneficial reference for the maximization of lignin waste valorization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Study of the mechanism of Flavobacterium sp. for hydrolyzing organophosphate pesticides.

    Science.gov (United States)

    Ortiz-Hernández, M L; Quintero-Ramírez, R; Nava-Ocampo, A A; Bello-Ramírez, A M

    2003-12-01

    The biotransformation by Flavobacterium sp. of the following organophosphate pesticides was experimentally and theoretically studied: phorate, tetrachlorvinphos, methyl-parathion, terbufos, trichloronate, ethoprophos, phosphamidon, fenitrothion, dimethoate and DEF. The Flavobacterium sp. ATCC 27551 strain bearing the organophosphate-degradation gene was used. Bacteria were incubated in the presence of each pesticide for a duration of 7 days. Parent pesticides were identified and quantified by means of a gas-chromatography mass spectrum system. Activity was considered as the amount (micromol) of each pesticide degraded by Flavobacterium sp. Also, structural parameters obtained by means of the CAChe program package for biomolecules, the reactivity index of phosphorus, of oxygen at the P = O function and of sulfur at the P = S function, and lipophilicity (log Poct) (ALOGPS v. 2.0) were obtained for each pesticide. Pesticides were hydrolyzed at the bond between phosphorous and the heteroatom, producing phosphoric acid and three metabolites. Enzymatic activity was significantly explained by the following multiple linear relationship: Enzymatic activity = 162.2 - 9.5(dihedral angle energy) - 25.0(Total energy) - 0.51(Molecular weight). Finally, a mechanism of Flavobacterium sp. to hydrolyze pesticides was proposed.

  9. Enhancing the Adhesive Strength of a Plywood Adhesive Developed from Hydrolyzed Specified Risk Materials

    Directory of Open Access Journals (Sweden)

    Birendra B. Adhikari

    2016-08-01

    Full Text Available The current production of wood composites relies mostly on formaldehyde-based adhesives such as urea formaldehyde (UF and phenol formaldehyde (PF resins. As these resins are produced from non-renewable resources, and there are some ongoing issues with possible health hazard due to formaldehyde emission from such products, the purpose of this research was to develop a formaldehyde-free plywood adhesive utilizing waste protein as a renewable feedstock. The feedstock for this work was specified risk material (SRM, which is currently being disposed of either by incineration or by landfilling. In this report, we describe a technology for utilization of SRM for the development of an environmentally friendly plywood adhesive. SRM was thermally hydrolyzed using a Canadian government-approved protocol, and the peptides were recovered from the hydrolyzate. The recovered peptides were chemically crosslinked with polyamidoamine-epichlorohydrin (PAE resin to develop an adhesive system for bonding of plywood specimens. The effects of crosslinking time, peptides/crosslinking agent ratio, and temperature of hot pressing of plywood specimens on the strength of formulated adhesives were investigated. Formulations containing as much as 78% (wt/wt peptides met the ASTM (American Society for Testing and Materials specifications of minimum dry and soaked shear strength requirement for UF resin type adhesives. Under the optimum conditions tested, the peptides–PAE resin-based formulations resulted in plywood specimens having comparable dry as well as soaked shear strength to that of commercial PF resin.

  10. Physical and chemical characterization of hydrolyzed Napier grass waste for biomass pellets

    Directory of Open Access Journals (Sweden)

    Duangkanok Tanangteerapong

    2017-09-01

    Full Text Available Utilizing agricultural waste for energy and to conserve the environment has been an active research area recently. In this study, Napier grass waste resulting from its hydrolysis with acids was pelletized and its use was studied as a biomass fuel. An investigation of its physical characteristics, including pellet dimensions and weight, was performed at a laboratory scale. Flat-faced pellets were obtained with enhanced stability necessary for transportation. Chemical characterization of the hydrolyzed waste included determination of its nitrogen, chloride and sulfur content. These values were marginal. After pelletization, experimental determination of its heating values, moisture content and density was examined. It was found that these values exceeded the European standard for biomass pellets. The average of heating value of pelletized biomass was greater than 3500 cal/g. Therefore, a binding material was not necessary. Overall, the investigation revealed that the pellets produced from hydrolyzed Napier grass waste could potentially be exploited as an alternative energy source after some residual chemicals were removed.

  11. ENGINEERING APPLIED TO FOOD PRODUCTION FORMULATED BY HYDROLYZED BAGASSE. A CASE OF STUDY

    Directory of Open Access Journals (Sweden)

    Raúl Costales Sotelo

    2015-01-01

    Full Text Available Some aspects of the engineering applied to the production of formulated food by hydrolyzed bagasse are showed, as an alternative in which fibrous component with increased digestibility constitutes 77% of the portion together with molasses at 15.9%, for almost 93% the total foodstuff potentially possible to be provided for a traditional sugar industry. The others ingredients such as urea, salts and minerals are common in animal diet and also in animal health and physiology. Local agriculture can contribute significantly to this program but is not taken into account in this exercise. This alternative, possible and feasible under Cuban economy conditions is magnified by the argument of operating periods of production facilities during and exceed normal sugar mill campaign for animals confinement periods of 180 days or longer, avoiding not only animals mortality but gaining weight at a rate of 500 g/day in a dry season by the input of 12 kg of formulated product per day. We have a first hydrolyzed plant which represents the beginning of an investment program that covers four more replicates, scattered throughout the national territory and it will significantly reduce food deficits needed by cattle as the main element of care, obtained by dual purpose: milk and meat production.

  12. In vitro study of partially hydrolyzed poly(2-ethyl-2-oxazolines) as materials for biomedical applications.

    Science.gov (United States)

    Shah, Rushita; Kronekova, Zuzana; Zahoranová, Anna; Roller, Ladislav; Saha, Nabanita; Saha, Petr; Kronek, Juraj

    2015-04-01

    Polymers based on 2-oxazoline, such as poly(2-ethyl-2-oxazolines) (PETOx), are considered to be a type of 'pseudopeptide' with the ability to form novel biomaterials. The hydrolysis of PETOx was carried out to evaluate its use in biomedical applications. In the present work, PETOx samples with a range of molar masses were prepared by living cationic polymerization. Hydrolysis was carried out at time intervals ranging from 15 to 180 min to prepare copolymers with different amounts of ethylene imine units. (1)H NMR spectroscopy was used to identify the structure of the hydrolyzed polymers. The dependence of in vitro cell viability on the degree of hydrolysis was determined using three different model cell lines, namely, mouse embryonic 3T3 fibroblasts, pancreatic βTC3 cells, and mouse lymphoid macrophages P388.D1. It was demonstrated that increasing the degree of hydrolysis decreased cell viability for all cell types. Fibroblast cells displayed the highest tolerance; additionally, the effect of polymer size showed no observable significance. Macrophage cells, immune system representatives, displayed the highest sensitivity to contact with hydrolyzed PETOx. The effect of polymer hydrolysis, polymer concentration and the incubation time on cell viability was experimentally observed. Confocal laser-scanning microscopy provided evidence of cellular uptake of pyrene-labeled (co)polymers.

  13. Bacillus thuringiensis fermentation of hydrolyzed sludge--rheology and formulation studies.

    Science.gov (United States)

    Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2007-03-01

    Rheology of Bacillus thuringiensis fermentation of hydrolyzed sludge was investigated in bench scale fermenter. Stable liquid formulations were developed and optimized for two-year based studies comprising various physical/chemical (viscosity, particle size, corrosion and suspendibility) and biological (microbial contamination, viable spores and entomotoxicity) parameters at different pHs and temperatures. The hydrolyzed sludge depicted non-Newtonian and pseudoplastic behaviour during fermentation with 90% to 96% confidence of fits into Casson, Power and IPC paste models. Higher values of consistency and flow index during exponential growth and stationary phase, respectively, affected downstream processing. The power law was also followed by stable formulations. Sorbitol, sodium monophosphate and sodium metabisulfite (2.2:1:1) as suspending agents produced suspendibility ranging from 69% to 94%. The stable formulation (FH-4) comprising sorbitol, sodium monophosphate and sodium metabisulfite deteriorated at pHs 6, 6.5 and temperatures, 40 and 50 degrees C, with no signs of corrosion and microbial contamination. The viscosity of FH-4 formulations decreased with shear rate which could improve handling and consequent spraying.

  14. Bioactive Peptides from Angelica sinensis Protein Hydrolyzate Delay Senescence in Caenorhabditis elegans through Antioxidant Activities

    Science.gov (United States)

    Wang, Qiangqiang; Huang, Yunxuan; Qin, Chuixin; Liang, Ming; Mao, Xinliang; Li, Shuiming; Zou, Yongdong; Jia, Weizhang; Li, Haifeng; Ma, Chung Wah; Huang, Zebo

    2016-01-01

    Since excessive reactive oxygen species (ROS) is known to be associated with aging and age-related diseases, strategies modulating ROS level and antioxidant defense systems may contribute to the delay of senescence. Here we show that the protein hydrolyzate from Angelica sinensis was capable of increasing oxidative survival of the model animal Caenorhabditis elegans intoxicated by paraquat. The hydrolyzate was then fractionated by ultrafiltration, and the antioxidant fraction (<3 kDa) was purified by gel filtration to obtain the antioxidant A. sinensis peptides (AsiPeps), which were mostly composed of peptides with <20 amino acid residues. Further studies demonstrate that AsiPeps were able to reduce the endogenous ROS level, increase the activities of the antioxidant enzymes superoxide dismutase and catalase, and decrease the content of the lipid peroxidation product malondialdehyde in nematodes treated with paraquat or undergoing senescence. AsiPeps were also shown to reduce age pigments accumulation and extend lifespan but did not affect the food-intake behavior of the nematodes. Taken together, our results demonstrate that A. sinensis peptides (AsiPeps) are able to delay aging process in C. elegans through antioxidant activities independent of dietary restriction. PMID:26941890

  15. Application of cross-linked and hydrolyzed arabinoxylans in baking of model rye bread.

    Science.gov (United States)

    Buksa, Krzysztof; Nowotna, Anna; Ziobro, Rafał

    2016-02-01

    The role of water extractable arabinoxylan with varying molar mass and structure (cross-linked vs. hydrolyzed) in the structure formation of rye bread was examined using a model bread. Instead of the normal flour, the dough contained starch, arabinoxylan and protein, which were isolated from rye wholemeal. It was observed that the applied mixes of these constituents result in a product closely resembling typical rye bread, even if arabinoxylan was modified (by cross-linking or hydrolysis). The levels of arabinoxylan required for bread preparation depended on its modification and mix composition. At 3% protein, the maximum applicable level of poorly soluble cross-linked arabinoxylan was 3%, as higher amounts of this preparation resulted in an extensively viscous dough and diminished bread volume. On the other hand highly soluble, hydrolyzed arabinoxylan could be used at a higher level (6%) together with larger amounts of rye protein (3% or 6%). Further addition of arabinoxylan leads to excessive water absorption, resulting in a decreased viscosity of the dough during baking and insufficient gas retention. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Simulated moving bed separation of agarose-hydrolyzate components for biofuel production from marine biomass.

    Science.gov (United States)

    Kim, Pung-Ho; Nam, Hee-Geun; Park, Chanhun; Wang, Nien-Hwa Linda; Chang, Yong Keun; Mun, Sungyong

    2015-08-07

    The economically-efficient separation of galactose, levulinic acid (LA), and 5-hydroxymethylfurfural (5-HMF) in acid hydrolyzate of agarose has been a key issue in the area of biofuel production from marine biomass. To address this issue, an optimal simulated moving bed (SMB) process for continuous separation of the three agarose-hydrolyzate components with high purities, high yields, and high throughput was developed in this study. As a first step for this task, the adsorption isotherm and mass-transfer parameters of each component on the qualified adsorbent were determined through a series of multiple frontal experiments. The determined parameters were then used in optimizing the SMB process for the considered separation. Finally, the optimized SMB process was tested experimentally using a self-assembled SMB unit with four zones. The SMB experimental results and the relevant computer simulations verified that the developed process in this study was quite successful in the economically-efficient separation of galactose, LA, and 5-HMF in a continuous mode with high purities and high yields. It is thus expected that the developed SMB process in this study will be able to serve as one of the trustworthy ways of improving the economic feasibility of biofuel production from marine biomass. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. [Gradient elevation of temperature startup experiment of thermophilic ASBR treating thermal-hydrolyzed sewage sludge].

    Science.gov (United States)

    Ouyang, Er-Ming; Wang, Wei; Long, Neng; Li, Huai

    2009-04-15

    Startup experiment was conducted for thermophilic anaerobic sequencing batch reactor (ASBR) treating thermal-hydrolyzed sewage sludge using the strategy of the step-wise temperature increment: 35 degrees C-->40 degrees C-->47 degrees C-->53 degrees C. The results showed that the first step-increase (from 35 degrees C to 40 degrees C) and final step-increase (from 47 degrees C to 53 degrees C) had only a slight effect on the digestion process. The second step-increase (from 40 degrees C to 47 degrees C) resulted in a severe disturbance: the biogas production, methane content, CODeffluent and microorganism all have strong disturbance. At the steady stage of thermophilic ASBR treating thermal-hydrolyzed sewage sludge, the average daily gas production, methane content, specific methane production (CH4/CODinfluent), TCOD removal rate and SCOD removal rate were 2.038 L/d, 72.0%, 188.8 mL/g, 63.8%, 83.3% respectively. The results of SEM and DGGE indicated that the dominant species are obviously different at early stage and steady stage.

  18. Selective capture of most celiac immunogenic peptides from hydrolyzed gluten proteins.

    Science.gov (United States)

    Moreno, María de Lourdes; Muñoz-Suano, Alba; López-Casado, Miguel Ángel; Torres, María Isabel; Sousa, Carolina; Cebolla, Ángel

    2016-08-15

    The available immunomethods for gluten quantitation could underestimate or overestimate the net immunoactivity of foods and beverages if the chosen analytical antibody is not specific to the relevant gluten immunogenic peptides (GIP). Accurate detection of the most active GIP is desirable to assess the potential celiac toxicity of food. We evaluated the capacity of the G12 monoclonal antibody for selectively depleting GIP in samples from two different gluteomes. Samples of hydrolyzed gliadin from wheat and a barley beer were used. The input (starting peptide digest of prolamins), the flow-through (unbound peptides), and the output (captured peptides) were analyzed by G12 and R5 competitive ELISA as well as by stimulation assays of T-cells from celiac patients. Most of the GIP were retained by the G12-agarose and represented the largest part of the immunogenicity of the gluten peptidome. G12 immunodepletion experiments with hydrolyzed gluten showed that this antibody reacted with those with the highest immunoactivity for celiac patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Effect Of Hydrolyzed Milk On The Adhesion Of Lactobacilli To Intestinal Cells*

    Directory of Open Access Journals (Sweden)

    Volštátová T.

    2015-03-01

    Full Text Available Milk is an essential part of the human diet and is undoubtedly a major calcium source in human nutrition, accepted well by most individuals. Knowledge on how the components from dairy products support or reduce the adherence of probiotics to the intestinal epithelium is limited. The purpose of this study was to investigate the effect of acid-hydrolyzed milk on the adhesion ability of two potentially probiotic strains (Lactobacillus plantarum S2, Lactobacillus gasseri R to in vitro human intestinal epithelial model consisting of Caco-2 and mucus-secreting HT29-MTX co-culture. The adhesion of our tested strains L. gasseri and L. plantarum was 4.74 and 7.16%, respectively, when using inoculum of 2 × 108 CFU ml–1. Addition of acid-hydrolyzed milk to co-culture decreased the adherence by 53.7% for L. gasseri R and by 62.2% for L. plantarum S2. The results of this study evidently indicate the potential importance of the food matrix as a factor influencing probiotic colonization of the gut.

  20. Growth promotion of Bifidobacterium species by poultry bone and meat trimming hydrolyzate.

    Science.gov (United States)

    Lazzi, Camilla; Meli, Federica; Dossena, Arnaldo; Gatti, Monica; Neviani, Erasmo

    2011-08-01

    The growth of bifidobacteria that are employed in the production of functional food is often slow or limited, even on synthetic media. In this study, we investigated whether a peptide hydrolyzate (functional animal protein [FAP]), from poultry bones and meat trimmings, could be a potential source of growth stimulators. The bifidogenic activity of FAP on 18 strains of Bifidobacterium species was assessed via 2 different techniques: turbidimetric measurements and a direct count by fluorescence microscopy. Growth experiments were performed in B12 broth as the basal medium, B12 broth supplemented with N-acetylglucosamine, and B12 broth supplemented with FAP. FAP supplementation yielded the highest maximum optical density (OD) and count values. The use of the microscopic fluorescence counts allowed for better evaluation of the extent of growth and assessment of the viability of cells. FAP from poultry bones and meat trimmings has potential as a growth stimulator for different bifidobacteria of human origin. FAP is a promising ingredient for inclusion in industrial media that are used to culture probiotic strains, including bifidobacteria, because it supports growth very well and maintains cells at a high level of viability. Proteinaceous hydrolyzate can be considered a promising ingredient for industrial media that are used to culture probiotic strains, including bifidobacteria, because it improves bacterial growth and maintains cells at a high level of viability. © 2011 Institute of Food Technologists®

  1. Costus afer Possesses Carbohydrate Hydrolyzing Enzymes Inhibitory Activity and Antioxidant Capacity In Vitro

    Directory of Open Access Journals (Sweden)

    Armelle D. Tchamgoue

    2015-01-01

    Full Text Available Diabetes mellitus is a metabolic disorder of glucose metabolism which correlates with postprandial hyperglycemia and oxidative stress. Control of blood glucose level is imperative in the management of diabetes. The present study tested the hypothesis that Costus afer, an antihyperglycemic medicinal plant, possesses inhibitory activity against carbohydrate hydrolyzing enzymes. Hexane, ethyl acetate, methanol, and water extracts were prepared from the leaf, stem, and rhizome of C. afer and subjected to phytochemical screening, assayed for α-amylase and α-glucosidase inhibitory activities and antioxidant capacity (determined by total phenolic and total flavonoids contents, ferric reducing antioxidant power (FRAP, and DPPH radical scavenging activity. All extracts inhibited α-amylase and α-glucosidase activities. Ethyl acetate rhizome and methanol leaf extracts exhibited the best inhibitory activity against α-amylase and α-glucosidase (IC50: 0.10 and 5.99 mg/mL, respectively. Kinetic analysis revealed two modes of enzyme inhibition (competitive and mixed. All extracts showed antioxidant capacity, with hexane extracts exhibiting the best activity. DPPH assay revealed that methanol leaf, rhizome, and ethyl acetate stem extracts (IC50 < 5 mg/mL were the best antioxidants. The presence of bioactive compounds such as flavonoids, alkaloids, phenols, and tannins may account for the antioxidant capacity and carbohydrate hydrolyzing enzyme inhibitory activity of C. afer.

  2. Lactose-hydrolyzed milk is more prone to chemical changes during storage than conventional ultra-high-temperature (UHT) milk.

    Science.gov (United States)

    Jansson, Therese; Clausen, Morten R; Sundekilde, Ulrik K; Eggers, Nina; Nyegaard, Steffen; Larsen, Lotte B; Ray, Colin; Sundgren, Anja; Andersen, Henrik J; Bertram, Hanne C

    2014-08-06

    The enzymatic hydrolysis of lactose to glucose and galactose gives rise to reactions that change the chemistry and quality of ambient-stored lactose-hydrolyzed ultra-high-temperature (UHT) milk. The aim of the present study was to investigate and compare chemical changes in lactose-hydrolyzed and conventional UHT milk during a 9 month ambient storage period. Several complementary analyses of volatiles, free amino acids, acetate, furosine, and level of free amino terminals were concluded. The analyses revealed an increased level of free amino acids and an increased formation rate of specific compounds such as furosine and 2-methylbutanal in lactose-hydrolyzed UHT milk compared to conventional UHT milk during storage. These observations indicate more favorable conditions for Maillard and subsequent reactions in lactose-hydrolyzed milk compared to conventional UHT milk stored at ambient temperature. Furthermore, it is postulated that proteolytic activity from the lactase-enzyme preparation may be responsible for the observed higher levels of free amino acids in lactose-hydrolyzed UHT milk.

  3. Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2008-12-01

    Full Text Available Abstract Background Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes and developing various biotechnological applications. Results The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45–49%. The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3

  4. ATP level and caffeine efficiency on cytokinesis inhibition in plants.

    Science.gov (United States)

    López-Sáez, J F; Mingo, R; González-Fernández, A

    1982-06-01

    Plant cytokinesis appears to be a topographically organized process of exocytosis. Golgi vesicles which contain cell wall precursors are translocated during telophase, by interzonal microtubules, to the equatorial region of the mitotic apparatus where they fuse with each other giving rise to the new cell wall. Caffeine inhibits cytokinesis by hindering Golgi vesicle coalescence. The present results demonstrate that treatments which increase the cellular ATP level (adenosine, cycloheximide and anisomycin) counteract caffein-induced cytokinesis inhibition in meristem cells of onion root tips (Allium cepa L.), while treatments which decrease ATP level potentiate this caffeine effect (dinitrophenol, fluoroacetate, low oxygen tensions, etc.). We postulate that caffeine, in competition with the cellular ATP level, blocks cell plate formation by inhibiting a certain ATPase activity required for membrane fusion of Golgi vesicles.

  5. 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...

  6. 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......) of the total ankle dorsiflexor muscle volume, which was 267 +/- 10 cm. Relative cross-sectional areas occupied by Type I, IIA, and IIB fibers in the tibialis anterior were 69.3 +/- 2.2, 27.4 +/- 2.76, and 3.2 +/- 1.0%, respectively. ATP economy of force maintenance did not change significantly during the 60-s...... contraction. It averaged at 4.81 +/- 0.42 N.s.micromol-1, and correlated with the relative cross-sectional area of the muscle occupied by Type I fiber (r = 0.73, P economy compared with those maintaining the force (3...

  7. Arabidopsis fructokinase-like protein associations are regulated by ATP.

    Science.gov (United States)

    Riggs, John W; Callis, Judy

    2017-05-10

    The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1-FLN2 hetero-oligomer is the dominant form in vitro We further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light-dark cycles in the 1-5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  8. EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

    Directory of Open Access Journals (Sweden)

    Olsén Arne

    2008-01-01

    Full Text Available Abstract Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. Results We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. Conclusion We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

  9. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

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    Alexis Díaz-Vegas

    Full Text Available During exercise, skeletal muscle produces reactive oxygen species (ROS via NADPH oxidase (NOX2 while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

  10. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive.

    Science.gov (United States)

    Calapez, Alexandre; Pereira, Henrique M; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C; Carmo-Fonseca, Maria

    2002-12-09

    After being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22 degrees C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.

  11. Students’ reasoning about “high-energy bonds” and ATP: A vision of interdisciplinary education

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    Benjamin W. Dreyfus

    2014-05-01

    Full Text Available As interdisciplinary courses are developed, instructors and researchers have to grapple with questions of how students should make connections across disciplines. We explore the issue of interdisciplinary reconciliation (IDR: how students reconcile seemingly contradictory ideas from different disciplines. While IDR has elements in common with other frameworks for the reconciliation of ideas across contexts, it differs in that each disciplinary idea is considered canonically correct within its own discipline. The setting for the research is an introductory physics course for biology majors that seeks to build greater interdisciplinary coherence and therefore includes biologically relevant topics such as adenosine triphosphate (ATP and chemical bond energy. In our case-study data, students grapple with the apparent contradiction between the energy released when the phosphate bond in ATP is broken and the idea that an energy input is required to break a bond. We see students justifying context-dependent modeling choices, showing nuance in articulating how system choices may be related to disciplinary problems of interest. This represents a desired end point of IDR, in which students can build coherent connections between concepts from different disciplines while understanding each concept in its own disciplinary context. Our case study also illustrates elements of the instructional environment that play roles in the process of IDR.

  12. ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis

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    Killen García

    2016-01-01

    Full Text Available Neisseria gonorrhoeae (Ngo has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM. Here, we investigate the role of adenosine triphosphate (ATP in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P0.05 and caspase-1 (CASP1, P>0.05. In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P>0.01. Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

  13. Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzing β-Lactamase KPC-2

    Science.gov (United States)

    Yigit, Hesna; Queenan, Anne Marie; Rasheed, J. Kamile; Biddle, James W.; Domenech-Sanchez, Antonio; Alberti, Sebastian; Bush, Karen; Tenover, Fred C.

    2003-01-01

    We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the blaSHV and blaTEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The blaKPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination. PMID:14638498

  14. Abnormal expression of ATP1A1 and ATP1A2 in breast cancer [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Alexey Bogdanov

    2017-01-01

    Full Text Available Breast cancer is the first in incidence and the second in death among all solid tumors occurring in women. The identification of molecular genetic abnormalities in breast cancer is important to improve the results of treatment. In the present study, we analyzed microarray data of breast cancer expression profiling (NCBI GEO database, accession GSE65194, focusing on Na+/K+-ATPase coding genes. We found overexpression of the ATP1A1 and down-regulation of the ATP1A2. We expect that our research could help to improve the understanding of predictive and prognostic features of breast cancer.

  15. Chemical Mechanism of the Phosphotriesterase from Sphingobium sp. Strain TCM1, an Enzyme Capable of Hydrolyzing Organophosphate Flame Retardants.

    Science.gov (United States)

    Bigley, Andrew N; Xiang, Dao Feng; Ren, Zhongjie; Xue, Haoran; Hull, Kenneth G; Romo, Daniel; Raushel, Frank M

    2016-03-09

    The mechanism of action of the manganese-dependent phosphotriesterase from Sphingobium sp. strain TCM1 that is capable of hydrolyzing organophosphate flame retardants was determined. The enzyme was shown to hydrolyze the RP-enantiomer of O-methyl O-cyclohexyl p-nitrophenyl thiophosphate with net inversion of configuration and without the formation of a covalent reaction intermediate. These results demonstrate that the enzyme catalyzes the hydrolysis of substrates by activation of a nucleophilic water molecule for direct attack at the phosphorus center.

  16. Bioluminometric assay of ATP in mouse brain: Determinant factors ...

    Indian Academy of Sciences (India)

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the ...

  17. 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.

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

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    The membrane-bound H+-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth...

  19. Two-step ATP-driven opening of cohesin head.

    Science.gov (United States)

    Marcos-Alcalde, Íñigo; Mendieta-Moreno, Jesús I; Puisac, Beatriz; Gil-Rodríguez, María Concepción; Hernández-Marcos, María; Soler-Polo, Diego; Ramos, Feliciano J; Ortega, José; Pié, Juan; Mendieta, Jesús; Gómez-Puertas, Paulino

    2017-06-12

    The cohesin ring is a protein complex composed of four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. It is involved in chromosome segregation, DNA repair, chromatin organization and transcription regulation. Opening of the ring occurs at the "head" structure, formed of the ATPase domains of Smc1A and Smc3 and Rad21. We investigate the mechanisms of the cohesin ring opening using techniques of free molecular dynamics (MD), steered MD and quantum mechanics/molecular mechanics MD (QM/MM MD). The study allows the thorough analysis of the opening events at the atomic scale: i) ATP hydrolysis at the Smc1A site, evaluating the role of the carboxy-terminal domain of Rad21 in the process; ii) the activation of the Smc3 site potentially mediated by the movement of specific amino acids; and iii) opening of the head domains after the two ATP hydrolysis events. Our study suggests that the cohesin ring opening is triggered by a sequential activation of the ATP sites in which ATP hydrolysis at the Smc1A site induces ATPase activity at the Smc3 site. Our analysis also provides an explanation for the effect of pathogenic variants related to cohesinopathies and cancer.

  20. Diversity and regulation of ATP sulfurylase in photosynthetic organisms

    Directory of Open Access Journals (Sweden)

    Laura ePrioretti

    2014-11-01

    Full Text Available ATP sulfurylase (ATPS catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper challenge this view and suggest that ATPSes may have a crucial regulatory role in sulfate assimilation, at least in algae.In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s expression. Special attention is given to algae ATPSes. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPSes revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. For instance, algae ATPSes show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. It is interesting that, at least with respect to the number of cysteines, the ATPSes of eukaryotic algae are closer to the marine cyanobacteria of the genera Synechococcus and Prochlorococcus and are more distant from freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater.

  1. Familial Hemiplegic Migraine With ATP1A2 Mutations

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2007-05-01

    Full Text Available Three children with prolonged hemiplegia following severe unilateral headache and having mutations in ATP1A2 are reported from UCLA School of Medicine, Los Angeles, CA; University Children’s Hospital, Zurich, Switzerland; and Wake Forest University School of Medicine, Winston-Salem, NC.

  2. Distinct neurological disorders with ATP1A3 mutations

    NARCIS (Netherlands)

    Heinzen, E.L.; Arzimanoglou, A.; Brashear, A.; Clapcote, S.J.; Gurrieri, F.; Goldstein, D.B; Johannesson, S.H.; Mikati, M.A.; Neville, B.; Nicole, S.; Ozelius, L.J.; Poulsen, H.; Schyns, T.; Sweadner, K.J.; Maagdenberg, A. van den; Vilsen, B.; Koenderink, J.B.

    2014-01-01

    Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the alpha3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological

  3. Abiogenic Photophosphorylation of ADP to ATP Sensitized by Flavoproteinoid Microspheres

    Science.gov (United States)

    Kolesnikov, Michael P.; Telegina, Taisiya A.; Lyudnikova, Tamara A.; Kritsky, Mikhail S.

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10 20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer left( {{text{FlH}}^ bullet } right) and ADP are involved.

  4. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  5. Hair bundles are specialized for ATP delivery via creatine kinase.

    NARCIS (Netherlands)

    Shin, J.B.; Streijger, F.; Beynon, A.J.; Peters, T.; Gadzala, L.; McMillen, D.; Bystrom, C.; Zee, C.E.E.M. van der; Wallimann, T.; Gillespie, P.G.

    2007-01-01

    When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to

  6. Treatment of heterotopic ossification through remote ATP hydrolysis.

    Science.gov (United States)

    Peterson, Jonathan R; De La Rosa, Sara; Eboda, Oluwatobi; Cilwa, Katherine E; Agarwal, Shailesh; Buchman, Steven R; Cederna, Paul S; Xi, Chuanwu; Morris, Michael D; Herndon, David N; Xiao, Wenzhong; Tompkins, Ronald G; Krebsbach, Paul H; Wang, Stewart C; Levi, Benjamin

    2014-09-24

    Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation. Copyright © 2014, American Association for the Advancement of Science.

  7. Clipboard: New paradigm for ATP synthesis and consumption

    Indian Academy of Sciences (India)

    2011-03-14

    Mar 14, 2011 ... Home; Journals; Journal of Biosciences; Volume 36; Issue 1. Clipboard: New paradigm for ATP synthesis and consumption. C Channakeshava. Volume 36 Issue 1 March 2011 pp 3-4. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/jbsc/036/01/0003-0004 ...

  8. Differential expression of ATP-dependent RNA helicase gene in ...

    African Journals Online (AJOL)

    AJL

    2012-02-07

    Feb 7, 2012 ... lysogeny broth (LB) medium at 4°C was induced for VBNC state; activated genes were detected using. mRNA differential display .... 191 amino acids. This cDNA sequence had a homology of 95 to 100% to the nucleotide of adenosine tripho- sphate (ATP)-dependent RNA helicase rh1B gene in different ...

  9. Cyclodextrin-based microcapsules as bioreactors for ATP biosynthesis.

    Science.gov (United States)

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

    2013-09-09

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

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

    Science.gov (United States)

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

    2014-11-01

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

  11. Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C.

    Science.gov (United States)

    Marzahn, Melissa R; Hayner, Jaclyn N; Meyer, Jennifer A; Bloom, Linda B

    2015-01-01

    DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the Saccharomyces cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Intercellular Odontoblast Communication via ATP Mediated by Pannexin-1 Channel and Phospholipase C-coupled Receptor Activation

    Science.gov (United States)

    Sato, Masaki; Furuya, Tadashi; Kimura, Maki; Kojima, Yuki; Tazaki, Masakazu; Sato, Toru; Shibukawa, Yoshiyuki

    2015-01-01

    Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected from rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s), we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i) by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK) 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP) release channel (pannexin-1) inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC) inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated odontoblast

  13. Intercellular odontoblast communication via ATP mediated by pannexin-1 channel and phospholipase C-coupled receptor activation.

    Directory of Open Access Journals (Sweden)

    Masaki eSato

    2015-11-01

    Full Text Available Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected form rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s, we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP release channel (pannexin-1 inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated

  14. Function and application of a non-ester-hydrolyzing carboxylesterase discovered in tulip.

    Science.gov (United States)

    Nomura, Taiji

    2017-01-01

    Plants have evolved secondary metabolite biosynthetic pathways of immense rich diversity. The genes encoding enzymes for secondary metabolite biosynthesis have evolved through gene duplication followed by neofunctionalization, thereby generating functional diversity. Emerging evidence demonstrates that some of those enzymes catalyze reactions entirely different from those usually catalyzed by other members of the same family; e.g. transacylation catalyzed by an enzyme similar to a hydrolytic enzyme. Tuliposide-converting enzyme (TCE), which we recently discovered from tulip, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. The TCEs belong to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme showing catalytic properties that are unpredictable from its primary structure. This review describes the biochemical and physiological aspects of tulipalin biogenesis, and the diverse functions of plant carboxylesterases in the α/β-hydrolase fold superfamily.

  15. Effects of dilute-acid pretreatment conditions on filtration performance of corn stover hydrolyzate.

    Science.gov (United States)

    Sievers, David A; Kuhn, Erik M; Tucker, Melvin P; McMillan, James D

    2017-11-01

    The reaction conditions used during dilute-acid pretreatment of lignocellulosic biomass control the carbohydrate digestion yield and also hydrolyzate properties. Depending on the conversion route of interest, solid-liquid separation (SLS) may be required to split the hemicellulose-rich liquor from the cellulose-rich insoluble solids, and slurry properties are important for SLS. Corn stover was pretreated at different reaction conditions and the slurries were assessed for conversion yield and filtration performance. Increasing pretreatment temperature reduced the solids mean particle size and resulted in slower slurry filtration rates when vacuum filtered or pressure filtered. Corn stover pretreated at 165°C for 10min and with 1% H2SO4 exhibited the highest xylose yield and best filtration performance with a no-wash filtration rate of 80kg/hm2 and cake permeability of 15x10-15. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Phenolic acids, hydrolyzable tannins, and antioxidant activity of geopropolis from the stingless bee Melipona fasciculata Smith.

    Science.gov (United States)

    Dutra, Richard Pereira; Abreu, Bruno Vinicius de Barros; Cunha, Mayara Soares; Batista, Marisa Cristina Aranha; Torres, Luce Maria Brandão; Nascimento, Flavia Raquel Fernandes; Ribeiro, Maria Nilce Sousa; Guerra, Rosane Nassar Meireles

    2014-03-26

    Geopropolis is a mixture of plant resins, waxes, and soil produced by the stingless bee Melipona fasciculata Smith. This paper describes the antioxidant activity and chemical composition of geopropolis produced by M. fasciculata. The total phenolic content determined with the Folin-Ciocalteu reagent was highest in the ethyl acetate fraction and hydroalcoholic extract. Antioxidant activity was assayed by the in vitro DPPH, ABTS, and FRAP assays. The hydroalcoholic extract and fractions of geopropolis, except for the hexane fraction, exhibited antioxidant activity against DPPH, ABTS, and FRAP. The phenolic compounds were identified by HPLC-DAD-MS on the basis of the evaluation of their UV-vis absorption maxima (λmax) and mass spectral analysis. Eleven compounds belonging to the classes of phenolic acids and hydrolyzable tannins (gallotannins and ellagitannins) were tentatively identified. These compounds are responsible for the antioxidant activity and high phenolic content of geopropolis produced by M. fasciculata.

  17. Antimicrobial and antioxidative activities of peptides from goat milk hydrolyzed with various protease

    Directory of Open Access Journals (Sweden)

    Eni Kusumaningtyas

    2015-09-01

    Full Text Available Milk is highly nutritious food containing protein as a good source of bioactive peptide that beneficial for health. This research was aimed to explore potency of bioactive peptide derived from goat milk as an antimicrobial and antioxidant. Milk was hydrolyzed by trypsin, chymotrypsin, pepsin, or protease Bacillus sp. E.13. The peptides obtained were screened for antimicrobial activities through incubation with Staphylococcus aureus, Listeria monocytogenes, Salmonella thyphimurium and Escherichia coli at 106 CFU/mL at 37°C for two hours and plated on Mueller Hinton agar. Antimicrobial activities were determined by comparing the total bacterial colonies to that of bacterial control without peptides addition. Oxidative activity was determined by 2.2’-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS and 2.2-diphenyl-1-picrylhydrazyl (DPPH assays. Antimicrobial activities were shown in peptides produced from hydrolysis of goat milk protein by pepsin at 37°C, pH 2 for 90 min and by Bacillus sp. E.13 protease at 55°C, pH 11 for 30 and 60 min but the activities were not detected in peptides from hydrolysis by trypsin and chymotrypsin. Peptide from protein hydrolysis by Bacillus sp. E.13 protease could inhibit Listeria monocytogenes, Salmonella thyphimurium and Escherichia coli up to 5 log cycles. The antimicrobial peptides could scavenge ABTS radical up to 86 % and DPPH radical up to 9 % at 68 μg protein/mL. Results indicated that goat milk protein hydrolyzed by Bacillus sp. E.13 protease is potential as antimicrobes and antioxidant.

  18. EFFECT OF ENDOSPERM HARDNESS ON AN ETHANOL PROCESS USING A GRANULAR STARCH HYDROLYZING ENZYME

    Energy Technology Data Exchange (ETDEWEB)

    Wang, P; W Liu, D B; Johnston, K D; Rausch, S J; Schmidt, M E; Tumbleson, V Singh

    2010-01-01

    Granular starch hydrolyzing enzymes (GSHE) can hydrolyze starch at low temperature (32°C). The dry grind process using GSHE (GSH process) has fewer unit operations and no changes in process conditions (pH 4.0 and 32°C) compared to the conventional process because it dispenses with the cooking and liquefaction step. In this study, the effects of endosperm hardness, protease, urea, and GSHE levels on GSH process were evaluated. Ground corn, soft endosperm, and hard endosperm were processed using two GSHE levels (0.1 and 0.4 mL per 100 g ground material) and four treatments of protease and urea addition. Soft and hard endosperm materials were obtained by grinding and sifting flaking grits from a dry milling pilot plant; classifications were confirmed using scanning electron microscopy. During 72 h of simultaneous granular starch hydrolysis and fermentation (GSHF), ethanol and glucose profiles were determined using HPLC. Soft endosperm resulted in higher final ethanol concentrations compared to ground corn or hard endosperm. Addition of urea increased final ethanol concentrations for soft and hard endosperm. Protease addition increased ethanol concentrations and fermentation rates for soft endosperm, hard endosperm, and ground corn. The effect of protease addition on ethanol concentrations and fermentation rates was most predominant for soft endosperm, less for hard endosperm, and least for ground corn. Samples (soft endosperm, hard endosperm, or corn) with protease resulted in higher (1.0% to 10.5% v/v) ethanol concentration compared to samples with urea. The GSH process with protease requires little or no urea addition. For fermentation of soft endosperm, GSHE dose can be reduced. Due to nutrients (lipids, minerals, and soluble proteins) present in corn that enhance yeast growth, ground corn fermented faster at the beginning than hard and soft endosperm.

  19. Activated sludge optimization using ATP in pulp and paper industry.

    Science.gov (United States)

    Bäckman, Göran; Gytel, Ulla

    2015-01-01

    The activated sludge process is an old technology, but still the most commonly used one for treatment of wastewater. Despite the wide spread usage the technology still suffers from instability (Tandoi et al. 2006) and high operating cost. Activated sludge processes often carry a large solids inventory. Managing the total inventory without interference is the key component of the optimization process described in this paper. Use of nutrients is common in pulp and paper effluent treatment. Feeding enough nutrients to support the biomass growth is a delicate balance. Overfeeding or underfeeding of nutrients can result in higher costs. Detrimental substances and toxic components in effluents entering a biological treatment system can cause severe, long lasting disturbances (Hynninen & Ingman 1998; Bergeron & Pelletier 2004). A LumiKem test kit is used to measure biological activity with adenosine triphosphate (ATP) in a pulp and paper mill. ATP data are integrated with other standardized mill parameters. Measurements of active volatile suspended solids based on ATP can be used to quantify the living biomass in the activated sludge process and to ensure that sufficient biomass is present in order to degrade the wastewater constituents entering the process. Information about active biomass will assist in optimizing sludge inventories and feeding of nutrients allowing the living biomass to re-populate to create optimal efficiency. ATP measurements can also be used to alert operators if any components toxic to bacteria are present in wastewater. The bio stress index represents the stress level experienced by the microbiological population. This parameter is very useful in monitoring toxicity in and around bioreactors. Results from the wastewater process optimization and ATP measurements showed that treatment cost could be reduced by approximately 20-30% with fewer disturbances and sustained biological activity compared to the reference period. This was mainly achieved by

  20. The influence of pH, temperature and hydrolyzate concentration on the removal of volatile and nonvolatile compounds from sugarcane bagasse hemicellulosic hydrolyzate treated with activated charcoal before or after vacuum evaporation

    Directory of Open Access Journals (Sweden)

    Rodrigues R.C.L.B.

    2001-01-01

    Full Text Available This paper analyzes the influence of pH, temperature and degree of hydrolyzate concentration on the removal of volatile and nonvolatile compounds from sugarcane bagasse hemicellulosic hydrolyzate treated with activated charcoal before or after the vacuum evaporation process. Furfural and 5-Hydroxymethylfurfural were almost totally removed in all the experiments, irrespective of pH and temperature and whether the charcoal was added before or after the vacuum evaporation process. Adding activated charcoal before the vacuum evaporation process favored the removal of phenolic compounds for all values of pH. Acetic acid, on the contrary, was most effectively removed when the activated charcoal was added after the vacuum evaporation process at an acid pH (0.92 and at the highest degree of hydrolyzate concentration (f=4. However, addition of activated charcoal before or after vacuum evaporation at an acid pH (0.92 and at the highest degree of hydrolyzate concentration (f=4 favored the removal of both acetic acid and phenolic compounds.

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

    Science.gov (United States)

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

    2003-01-01

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

  2. 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.

  3. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    Science.gov (United States)

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Wound-induced Ca2+wave propagates through a simple release and diffusion mechanism.

    Science.gov (United States)

    Handly, L Naomi; Wollman, Roy

    2017-06-01

    Damage-associated molecular patterns (DAMPs) are critical mediators of information concerning tissue damage from damaged cells to neighboring healthy cells. ATP acts as an effective DAMP when released into extracellular space from damaged cells. Extracellular ATP receptors monitor tissue damage and activate a Ca 2+ wave in the surrounding healthy cells. How the Ca 2+ wave propagates through cells after a wound is unclear. Ca 2+ wave activation can occur extracellularly via external receptors or intracellularly through GAP junctions. Three potential mechanisms to propagate the Ca 2+ wave are source and sink, amplifying wave, and release and diffusion. Both source and sink and amplifying wave regulate ATP levels using hydrolysis or secretion, respectively, whereas release and diffusion relies on dilution. Here we systematically test these hypotheses using a microfluidics assay to mechanically wound an epithelial monolayer in combination with direct manipulation of ATP hydrolysis and release. We show that a release and diffusion model sufficiently explains Ca 2+ -wave propagation after an epithelial wound. A release and diffusion model combines the benefits of fast activation at short length scales with a self-limiting response to prevent unnecessary inflammatory responses harmful to the organism. © 2017 Handly 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).

  5. Respiratory infections cause the release of extracellular vesicles: implications in exacerbation of asthma/COPD.

    Directory of Open Access Journals (Sweden)

    Suffwan Eltom

    Full Text Available Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations.To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis.Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1β/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia.This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

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

    Directory of Open Access Journals (Sweden)

    Jonathon eTelianidis

    2013-08-01

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

  7. Hydrolysis of native and heat-treated starches at sub-gelatinization temperature using granular starch hydrolyzing enzyme.

    Science.gov (United States)

    Uthumporn, U; Shariffa, Y N; Karim, A A

    2012-03-01

    The effect of heat treatment below the gelatinization temperature on the susceptibility of corn, mung bean, sago, and potato starches towards granular starch hydrolysis (35°C) was investigated. Starches were hydrolyzed in granular state and after heat treatment (50°C for 30 min) by using granular starch hydrolyzing enzyme for 24 h. Hydrolyzed heat-treated starches showed a significant increase in the percentage of dextrose equivalent compared to native starches, respectively, with corn 53% to 56%, mung bean 36% to 47%, sago 15% to 26%, and potato 12% to 15%. Scanning electron microscopy micrographs showed the presence of more porous granules and surface erosion in heat-treated starch compared to native starch. X-ray analysis showed no changes but with sharper peaks for all the starches, suggested that hydrolysis occurred on the amorphous region. The amylose content and swelling power of heat-treated starches was markedly altered after hydrolysis. Evidently, this enzyme was able to hydrolyze granular starches and heat treatment before hydrolysis significantly increased the degree of hydrolysis.

  8. Kinetic properties of two Rhizopus exo-polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry

    Science.gov (United States)

    The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a Km of 55 uM and kcat of 10.3 s^-1^ while RPG16 was shown to have greater af...

  9. Structure, function, and evolution of bacterial ATP-binding cassette systems

    Energy Technology Data Exchange (ETDEWEB)

    Davidson, A.L.; Dassa, E.; Orelle, C.; Chen, J. (Purdue)

    2010-07-27

    The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of a nucleotide-binding site, and the signature motif, unique to ABC proteins, located upstream of the Walker B motif (426). Other motifs diagnostic of ABC proteins are also indicated in Fig. 1. The biological significance of these motifs is discussed in Structure, Function, and Dynamics of the ABC. ABC systems are widespread among living organisms and have been detected in all genera of the three kingdoms of life, with remarkable conservation in the primary sequence of the cassette and in the organization of the constitutive domains or subunits (203, 420). ABC systems couple the energy of ATP hydrolysis to an impressively large variety of essential biological phenomena, comprising not only transmembrane (TM) transport, for which they are best known, but also several non-transport-related processes, such as translation elongation (62) and DNA repair (174). Although ABC systems deserve much attention because they are involved in severe human inherited diseases (107), they were first discovered and characterized in detail in prokaryotes, as early as the 1970s (13, 148, 238, 468). The most extensively analyzed systems were the high-affinity histidine and maltose uptake systems of Salmonella enterica serovar Typhimurium and Escherichia coli. Over 2 decades ago, after the completion of the nucleotide sequences encoding these transporters in the respective laboratories of Giovanna Ames and Maurice Hofnung, Hiroshi Nikaido and colleagues noticed that the two systems displayed a global similarity in the nature of their components and, moreover, that the primary sequences of MalK and

  10. 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

  11. Partially hydrolyzed whey proteins prevent clinical symptoms in a cow's milk allergy mouse model and enhance regulatory T and B cell frequencies

    NARCIS (Netherlands)

    Kiewiet, Mensiena B Gea; van Esch, Betty C A M|info:eu-repo/dai/nl/304839256; Garssen, Johan|info:eu-repo/dai/nl/086369962; Faas, Marijke M; Vos, Paul

    2017-01-01

    SCOPE: Partially hydrolyzed cow's milk proteins are used to prevent cow's milk allergy in children. Here we studied the immunomodulatory mechanisms of partial cow's milk hydrolysates in vivo. METHODS AND RESULTS: Mice were sensitized with whey or partially hydrolyzed whey using cholera toxin.

  12. Partially hydrolyzed whey proteins prevent clinical symptoms in a cow's milk allergy mouse model and enhance regulatory T and B cell frequencies

    NARCIS (Netherlands)

    Kiewiet, Mensiena B. Gea; van Esch, Betty C. A. M.; Garssen, Johan; Faas, Marijke M.; de Vos, Paul

    2017-01-01

    Scope: Partially hydrolyzed cow's milk proteins are used to prevent cow's milk allergy in children. Here we studied the immunomodulatory mechanisms of partial cow's milk hydrolysates in vivo. Methods and results: Mice were sensitized with whey or partially hydrolyzed whey using cholera toxin.

  13. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph

    2017-01-01

    Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened......G or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin......, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards...

  14. Studies towards the synthesis of ATP analogs as potential glutamine synthetase inhibitors

    CSIR Research Space (South Africa)

    Salisu, S

    2011-05-01

    Full Text Available In research directed at the development of adenine triphosphate (ATP) analogs as potential glutamine synthetase (GS) inhibitors, adenine and allopurinol derivatives have been synthesized either as novel ATP analogs or as scaffolds...

  15. Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

    OpenAIRE

    Tomomi Ando; Hiromi Imamura; Ryosuke Suzuki; Hideki Aizaki; Toshiki Watanabe; Takaji Wakita; Tetsuro Suzuki

    2012-01-01

    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 energi...

  16. 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

    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...... 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...... 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....

  17. Controlled release of small molecules from silica xerogel with limited nanoporosity.

    Science.gov (United States)

    Chen, Rong; Qu, Haibo; Agrawal, Ashwin; Guo, Shaoyun; Ducheyne, Paul

    2013-01-01

    Conventional sol-gel processing requires several distinct steps involving hydrolysis, condensation and drying to obtain a highly porous, glassy solid material. With the goal of achieving controlled release of small molecules, herein we focus on the acceleration of the condensation and drying steps by casting the hydrolyzed sol on a large open surface to achieve a denser 100 % silica xerogel structure. Thus, cast xerogel with a more limited porosity was prepared. The effect of synthesis parameters during sol-gel synthesis on the release kinetics of bupivacaine, vancomycin and cephalexin was investigated. The release kinetics fitted well with the Higuchi model, suggesting a diffusional release mechanism. Combining the release and nanostructure data, the formation mechanism of cast xerogel is described. Without introducing additional precursors or additives into sol-gel systems, sol-gel casting is an easy technique that further expands the applicability of sol-gel materials as excellent carriers for the controlled release of a variety of drugs.

  18. 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

  19. 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 ...

  20. A rapid and convenient method for preparing salt-free (. gamma. -/sup 32/P)ATP

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, J.L.; Avruch, J.

    1981-09-15

    (..gamma..-/sup 32/P)ATP is prepared by an existing enzymatic method that yields approximately 95% incorporation of /sup 32/P into ATP. A rapid and convenient method for purifying the (..gamma..-/sup 32/P)ATP which results in a product free of both salt and buffer is reported.

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

    DEFF Research Database (Denmark)

    Cappeln, Gertrud; Jessen, Flemming

    2001-01-01

    Glycolysis was shown to occur during freezing of cod of decrease in glycogen and an increase in lactate. In addition, the ATP content decreased during freezing. Synthesis of ATP was measured as degradation of glycogen. During storage at -9 and - 12 degreesC it was found that degradation of ATP...

  2. 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...... intracortical inhibition (SICI), intracortical facilitation (ICF), short latency afferent inhibition (SAI) as markers of intracortical intrahemispheric inhibition/facilitation and the ipsilateral silent period (iSP) and paired-pulse interhemispheric inhibition (IHI) to probe interhemispheric motor interactions...

  3. Kinetics of signaling-DNA-aptamer-ATP binding

    Science.gov (United States)

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

    2009-03-01

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

  4. ATP Induces Disruption of Tight Junction Proteins via IL-1 Beta-Dependent MMP-9 Activation of Human Blood-Brain Barrier In Vitro

    Directory of Open Access Journals (Sweden)

    Fuxing Yang

    2016-01-01

    Full Text Available Disruption of blood-brain barrier (BBB follows brain trauma or central nervous system (CNS stress. However, the mechanisms leading to this process or the underlying neural plasticity are not clearly known. We hypothesized that ATP/P2X7R signaling regulates the integrity of BBB. Activation of P2X7 receptor (P2X7R by ATP induces the release of interleukin-1β (IL-1β, which in turn enhances the activity of matrix metalloproteinase-9 (MMP-9. Degradation of tight junction proteins (TJPs such as ZO-1 and occludin occurs, which finally contributes to disruption of BBB. A contact coculture system using human astrocytes and hCMEC/D3, an immortalized human brain endothelial cell line, was used to mimic BBB in vitro. Permeability was used to evaluate changes in the integrity of TJPs. ELISA, Western blot, and immunofluorescent staining procedures were used. Our data demonstrated that exposure to the photoreactive ATP analog, 3′-O-(4-benzoylbenzoyl adenosine 5′-triphosphate (BzATP, induced a significant decrease in ZO-1 and occludin expression. Meanwhile, the decrease of ZO-1 and occludin was significantly attenuated by P2X7R inhibitors, as well as IL-1R and MMP antagonists. Further, the induction of IL-1β and MMP-9 was closely linked to ATP/P2X7R-associated BBB leakage. In conclusion, our study explored the mechanism of ATP/P2X7R signaling in the disruption of BBB following brain trauma/stress injury, especially focusing on the relationship with IL-1β and MMP-9.

  5. Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer.

    Science.gov (United States)

    Li, Yapiao; Sun, Linlin; Zhao, Qiang

    2017-11-01

    We developed an aptamer-based competitive fluorescence anisotropy (FA)/fluorescence polarization (FP) assay for adenosine triphosphate (ATP). Different from the traditional fluorescence polarization immunoassays for small molecules, here DNA aptamer against ATP was used as affinity ligand, and tetramethylrhodamine (TMR) labeled ATP served as fluorescent tracer. The binding between TMR-labeled ATP and aptamer gave large FA due to molecular volume increase and restricted rotation of the dye-labeled ATP. When ATP was added in solution, ATP competitively displaced the TMR-labeled ATP from aptamer affinity complex, causing decrease of FA of TMR-labeled ATP. The buffer containing MgCl 2 and incubation at low temperature were preferred for large FA change in the FA assay. The FA change was further enhanced in this competitive FA assay by increasing the molecular weight of aptamer through extension of aptamer sequences or conjugating streptavidin protein on aptamer. This method allowed for the detection of ATP in the range from 0.5μM to 1mM, generating the maximum FA change about 0.187 (corresponding maximum FP change about 0.242). The detection of ATP spiked in diluted urine or serum sample was achieved, showing capability for analysis in complex sample matrix. This assay also enabled the detection of the analogues of ATP, e.g. adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP) with similar sensitivity. This aptamer-based competitive FA assay takes advantages of aptamer in ease of synthesis, good thermal stability, and facile modulating the molecular mass of aptamer. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

    Science.gov (United States)

    Nath, Sunil

    2016-12-01

    As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Alternative mitochondrial functions in cell physiopathology: beyond ATP production

    Directory of Open Access Journals (Sweden)

    Kowaltowski A.J.

    2000-01-01

    Full Text Available It is well known that mitochondria are the main site for ATP generation within most tissues. However, mitochondria also participate in a surprising number of alternative activities, including intracellular Ca2+ regulation, thermogenesis and the control of apoptosis. In addition, mitochondria are the main cellular generators of reactive oxygen species, and may trigger necrotic cell death under conditions of oxidative stress. This review concentrates on these alternative mitochondrial functions, and their role in cell physiopathology.

  8. Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction.

    Science.gov (United States)

    Shibukawa, Yoshiyuki; Sato, Masaki; Kimura, Maki; Sobhan, Ubaidus; Shimada, Miyuki; Nishiyama, Akihiro; Kawaguchi, Aya; Soya, Manabu; Kuroda, Hidetaka; Katakura, Akira; Ichinohe, Tatsuya; Tazaki, Masakazu

    2015-04-01

    Various stimuli induce pain when applied to the surface of exposed dentin. However, the mechanisms underlying dentinal pain remain unclear. We investigated intercellular signal transduction between odontoblasts and trigeminal ganglion (TG) neurons following direct mechanical stimulation of odontoblasts. Mechanical stimulation of single odontoblasts increased the intracellular free calcium concentration ([Ca(2+)]i) by activating the mechanosensitive-transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV4, and TRPA1, but not TRPM8 channels. In cocultures of odontoblasts and TG neurons, increases in [Ca(2+)]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca(2+)]i were abolished in the absence of extracellular Ca(2+) and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca(2+)]i in neighboring odontoblasts and TG neurons, but not in the stimulated odontoblasts. G-protein-coupled P2Y nucleotide receptor antagonists also inhibited the increases in [Ca(2+)]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca(2+)]i in neighboring TG neurons, but not in stimulated or neighboring odontoblasts. During mechanical stimulation of single odontoblasts, a connexin-43 blocker did not have any effects on the [Ca(2+)]i responses observed in any of the cells. These results indicate that ATP, released from mechanically stimulated odontoblasts via pannexin-1 in response to TRP channel activation, transmits a signal to P2X3 receptors on TG neurons. We suggest that odontoblasts are sensory receptor cells and that ATP released from odontoblasts functions as a neurotransmitter in the sensory transduction sequence for dentinal pain.

  9. [The sensitivity and clinical course of patients with wheat-dependent exercise-induced anaphylaxis sensitized to hydrolyzed wheat protein in facial soap].

    Science.gov (United States)

    Hiragun, Makiko; Ishii, Kaori; Hiragun, Takaaki; Shindo, Hajime; Mihara, Shoji; Matsuo, Hiroaki; Hide, Michihiro

    2011-12-01

    Recently an increasing number of patients with wheat-dependent exercise-induced anaphylaxis (WDEIA), developed during or after using hydrolyzed wheat protein (HWP)-containing soap (HWP-WDEIA), were reported in Japan. To clarify the relation between WDEIA and HWP-containing soap and their prognosis, we investigated the patients who visited Hiroshima University Hospital and were diagnosed as WDEIA from January 2010 to June 2011. We took detailed clinical histories, performed skin prick tests, serum immunoassays for antigen-specific IgE and basophil histamine release test, and followed up their clinical courses after the diagnosis. Among 36 patients with WDEIA, 30 patients had used only one type of HWP-soap. The patients with HWP-WDEIA were mainly women and had developed facial symptoms and angioedema. They suffered from blood pressure reductions less frequently than patients with conventional WDEIA. The levels of glutens-specific IgE were higher than those of ω-5 gliadin in patients with HWP-WDEIA (psoap. The development of HWP-WDEIA is associated with the use of HWP-soap. The sensitivities to HWP that cross reacts with non-processed wheat may be reduced or possibly cured after the discontinuation of HWP-soap.

  10. The sensitivity and clinical course of patients with wheat-dependent exercise-induced anaphylaxis sensitized to hydrolyzed wheat protein in facial soap - secondary publication.

    Science.gov (United States)

    Hiragun, Makiko; Ishii, Kaori; Hiragun, Takaaki; Shindo, Hajime; Mihara, Shoji; Matsuo, Hiroaki; Hide, Michihiro

    2013-09-01

    Recently, an increasing number of patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) have been reported in Japan. Most of them had developed this condition during or after using hydrolyzed wheat protein (HWP)-containing soap (HWP-WDEIA). To clarify the relation between WDEIA and HWP-containing soap and their prognosis, we retrospectively studied the patients who visited Hiroshima University Hospital and were diagnosed as WDEIA from January 2010 to June 2011. We took detailed clinical histories, performed skin prick tests, serum immunoassays for antigen-specific IgE and basophil histamine release test, and followed up their clinical courses after the diagnosis. Among 36 patients with WDEIA, 30 patients had used only one type of HWP-soap. The patients with HWP-WDEIA were mainly women and had developed facial symptoms and angioedema. They suffered from blood pressure reductions less frequently than patients with conventional WDEIA. The levels of gluten-specific IgE were higher than those of omega-5 gliadin in patients with HWP-WDEIA (P soap. The development of HWP-WDEIA is associated with the use of HWP-soap. The sensitivity to HWP that cross reacts with non-processed wheat may be reduced or possibly cured after the discontinuation of HWP-soap.

  11. Masked trichothecene and zearalenone mycotoxins withstand digestion and absorption in the upper GI tract but are efficiently hydrolyzed by human gut microbiota in vitro.

    Science.gov (United States)

    Gratz, Silvia W; Dinesh, Reshma; Yoshinari, Tomoya; Holtrop, Grietje; Richardson, Anthony J; Duncan, Gary; MacDonald, Susan; Lloyd, Antony; Tarbin, Jonathan

    2017-04-01

    Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and β-zearalenol) in the human gut in vitro. Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from five donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered as parent mycotoxins whereas 40-70% of zearalenone compounds were further metabolized to unknown metabolites. Our results demonstrate that masked trichothecenes will reach the colon intact to be released as parent mycotoxins by gut microbiota, hence contributing to mycotoxin exposure. Masked zearalenone compounds are metabolized by gut microbiota and epithelial cells and the identity and toxicity of metabolites remain to be determined. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

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

  13. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  14. ATP-regenerating system in the cilia of Paramecium caudatum.

    Science.gov (United States)

    Noguchi, M; Sawada, T; Akazawa, T

    2001-03-01

    The energy supply for eukaryotic ciliary and flagellar movement is thought to be maintained by ATP-regenerating enzymes such as adenylate kinase, creatine kinase and arginine kinase. In this study, the energy-supplying system for the ciliary movement of Paramecium caudatum was examined. Arginine kinase and adenylate kinase activities were detected in the cilia. To demonstrate that phosphoarginine satisfactorily supplies high-energy phosphate compounds into the narrow ciliary space, we prepared an intact ciliated cortical sheet from live Paramecium caudatum. These cortical sheets, with an intact ciliary membrane, produced a half-closed system in which each cilium was covered with a ciliary membrane with an opening to the cell body. Ciliary beating on the intact cortical sheets was induced by perfusing not only ATP but also ADP. Addition of phosphoarginine (0.2 mmol l(-1)) increased the beat frequency. A further increase in beat frequency was observed in 0.4 mmol l(-1) phosphoarginine, and this was enhanced when the cilia were reactivated with relatively low concentrations of ATP. We have demonstrated that phosphoarginine supplies energy as a 'phosphagen' for ciliary beating in Paramecium caudatum, suggesting that phosphoarginine functions not only as a reservoir of energy but also as a transporter of energy in these continuously energy-consuming circumstances. http://www.biologists.com/JEB/movies/jeb3123.html

  15. Adenosine Triphosphate-Triggered Release of Macromolecular and Nanoparticle Loads from Aptamer/DNA-Cross-Linked Microcapsules.

    Science.gov (United States)

    Liao, Wei-Ching; Lu, Chun-Hua; Hartmann, Raimo; Wang, Fuan; Sohn, Yang Sung; Parak, Wolfgang J; Willner, Itamar

    2015-09-22

    The synthesis of stimuli-responsive DNA microcapsules acting as carriers for different payloads, and being dissociated through the formation of aptamer-ligand complexes is described. Specifically, stimuli-responsive anti-adenosine triphosphate (ATP) aptamer-cross-linked DNA-stabilized microcapsules loaded with tetramethylrhodamine-modified dextran (TMR-D), CdSe/ZnS quantum dots (QDs), or microperoxidase-11 (MP-11) are presented. In the presence of ATP as trigger, the microcapsules are dissociated through the formation of aptamer-ATP complexes, resulting in the release of the respective loads. Selective unlocking of the capsules is demonstrated, and CTP, GTP, or TTP do not unlock the pores. The ATP-triggered release of MP-11 from the microcapsules enables the MP-11-catalyzed oxidation of Amplex UltraRed by H2O2 to the fluorescent product resorufin.

  16. Inhibition by amiloride and by Na-depletion of A23187-stimulated arachidonic acid and histamine release from rat mast cells

    DEFF Research Database (Denmark)

    Linnebjerg, H.; Hansen, Harald S.; Jensen, B.

    1988-01-01

    of protein kinase C inhibitors resulted in only a modest decrease of [C]arachidonic acid and histamine release. [C]Arachidonic acid was hydrolyzed from both phosphatidylcholine and triacylglycerol. Amiloride inhibited only the hydrolysis from phosphatidylcholine. It is suggested that an Na/H exchange...

  17. 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

  18. Performance of Partially-Hydrolyzed Polyacrylamide in Water Based Drilling Fluids

    Directory of Open Access Journals (Sweden)

    Ali Reza Nasiri*

    2013-05-01

    Full Text Available Fluid properties with constant improvement in efficiency have been noticeable as important criteria in drilling operation. The main drilling fluid properties highly depend on utilization of new polymers with high efficiency in drilling fluid composition. In this paper, the performance of a new polymer, called partially hydrolyzed polyacrylamide polymer (PHPA, is studied which has recently entered the drilling fluids industry in Iran. Hence viscosity property, fluid loss control and shale inhibition of this polymer have been evaluated based on an international standard method of API-13-I by considering the drilling and operational priorities of thecountry. Then the thermal effect, salt contaminants such as sodium chloride, calcium chloride, magnesium chloride and pH tolerance effect as major pollution indicators are also investigated in relation to polymeric fluid properties. The results obtained by the tests show that furthermore polymer PHPA increases rheological properties (apparent viscosity, plastic fluidity and yield point and it plays important role in increases in fluid loss. This polymer has also demonstrated acceptable resistance toward sodium chloride contaminants, but its efficiency decreases toward calcium and magnesium ion contaminants. The thermal tests show that polymer PHPA has high thermal stability up to 150°C. This polymer improves shale inhibition property and by encapsulation mechanism prevents dispersion of shale cuttings into the drilling fluid system as it stops any changes in fluid properties which will finally results inwellbore stability.

  19. Molecular characterization and in vitro digestibility of normal maize starch hydrolyzed by maltotriohydrolase.

    Science.gov (United States)

    Wu, Chunsen; Zhou, Xing; Wei, Benxi; Li, Hongyan; Tian, Yaoqi; Ali, Barkat; Xu, Xueming; Jin, Zhengyu

    2015-03-01

    Normal maize starch was hydrolyzed by the glucan 1,4-alpha-maltotriohydrolase (AMTS), and the changes in molecular characteristics and digestibility of starch were evaluated. Upon hydrolysis, maltotriose purity could be modified via controlled AMTS action. The transglycosylation of AMTS possibly happened during the extensive hydrolysis of starch. No single linear association between the z-average radius of gyration (Rz), conformation exponent (ν), apparent molecular density (ρ) and weight average molar mass (Mw) of the starch molecules could be established in the entire process of AMTS hydrolysis. Under mild hydrolysis (≤240 min), Rz and ρ displayed linear relationships with Mw. However, transitions of ν, Rz and ρ appeared after extensive hydrolysis (>240 min), due to the increase in the amount of short chains [degree of polymerization (DP)≤5]. The spherical starch molecule tends toward less compact and a structure between sphere and random coil after extensive hydrolysis. And the increase in the amount of DP≤12 chains and reduction of molecular dimension after AMTS hydrolysis restrict the digestibility of starch. The results of this study suggest that normal maize starch can be modulated by AMTS to produce the desired maltotriose syrup, starch molecular characteristics, and starch digestibility. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Wheat-Dependent Exercise-Induced Anaphylaxis Sensitized with Hydrolyzed Wheat Protein in Soap

    Directory of Open Access Journals (Sweden)

    Yuko Chinuki

    2012-01-01

    Full Text Available Wheat-dependent exercise-induced anaphylaxis (WDEIA is a specific form of wheat allergy typically induced by exercise after ingestion of wheat products. Wheat ω-5 gliadin is a major allergen associated with conventional WDEIA, and detection of serum immunoglobulin E (IgE specific to recombinant ω-5 gliadin is a reliable method for its diagnosis. Recently, an increased incidence of a new subtype of WDEIA, which is likely to be sensitized via a percutaneous and/or rhinoconjunctival route to hydrolyzed wheat protein (HWP, has been observed. All of the patients with this new subtype had used the same brand of soap, which contained HWP. Approximately half of these patients developed contact allergy several months later and subsequently developed WDEIA. In each of these patients, contact allergy with soap exposure preceded food ingestion-induced reactions. Other patients directly developed generalized symptoms upon ingestion of wheat products. The predominant observed symptom of the new WDEIA subtype was angioedema of the eyelids; a number of patients developed anaphylaxis. This new subtype of WDEIA has little serum ω-5 gliadin-specific serum IgE.

  1. Wheat-dependent exercise-induced anaphylaxis sensitized with hydrolyzed wheat protein in soap.

    Science.gov (United States)

    Chinuki, Yuko; Morita, Eishin

    2012-12-01

    Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a specific form of wheat allergy typically induced by exercise after ingestion of wheat products. Wheat ω-5 gliadin is a major allergen associated with conventional WDEIA, and detection of serum immunoglobulin E (IgE) specific to recombinant ω-5 gliadin is a reliable method for its diagnosis. Recently, an increased incidence of a new subtype of WDEIA, which is likely to be sensitized via a percutaneous and/or rhinoconjunctival route to hydrolyzed wheat protein (HWP), has been observed. All of the patients with this new subtype had used the same brand of soap, which contained HWP. Approximately half of these patients developed contact allergy several months later and subsequently developed WDEIA. In each of these patients, contact allergy with soap exposure preceded food ingestion-induced reactions. Other patients directly developed generalized symptoms upon ingestion of wheat products. The predominant observed symptom of the new WDEIA subtype was angioedema of the eyelids; a number of patients developed anaphylaxis. This new subtype of WDEIA has little serum ω-5 gliadin-specific serum IgE.

  2. Acetylcholine-hydrolyzing activities in soluble brain fraction: Characterization with reversible and irreversible inhibitors.

    Science.gov (United States)

    Estévez, Jorge; Selva, Verónica; Benabent, Mónica; Mangas, Iris; Sogorb, Miguel Ángel; Vilanova, Eugenio

    2016-11-25

    Some effects of organophosphorus compounds (OPs) esters cannot be explained through actions on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In soluble chicken brain fraction, three components (Eα, Eβ and Eγ) of pheny lvalerate esterase activity (PVase) were kinetically discriminated and their relationship with acetylcholine-hydrolyzing activity (cholinesterase activity) were studied in previous works. In this work, four enzymatic components (CS1, CS2, CS3 and CS4) of cholinesterase activity have been discriminated in soluble fraction, according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA and to reversible inhibitors ethopropazine and BW284C51. Cholinesterase component CS1 can be related to the Eα component of PVase activity and identified as butyrylcholinesterase (BuChE). No association and similarities can be stablished among the other PVase component (Eβ and Eγ) with the other cholinesterase components (CS2, CS3, CS4). The kinetic analysis has allowed us to stablish a method for discriminating the enzymatic component based on a simple test with two inhibitors. It can be used as biomarker in toxicological studies and for monitoring these cholinesterase components during isolation and molecular identification processes, which will allow OP toxicity to be understood by a multi-target approach. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Dissolved total hydrolyzable enantiomeric amino acids in precipitation: Implications on bacterial contributions to atmospheric organic matter

    Science.gov (United States)

    Yan, Ge; Kim, Guebuem; Kim, Jeonghyun; Jeong, Yu-Sik; Kim, Young Il

    2015-03-01

    We analyzed dissolved organic carbon (DOC), dissolved organic nitrogen (DON), and dissolved enantiomeric amino acids in precipitation samples collected at two sites in Korea over a one-year period. The average concentrations of DOC, DON, and total hydrolyzable amino acids at Seoul (an inland urban area) were lower than those at Uljin (a coastal rural area). The different bulk compositions of dissolved organic matter (DOM) at these two sites (reflected by qualitative indicators) were mainly attributed to differences in contributing sources. The D-enantiomers of four individual amino acids (aspartic acid, glutamic acid, serine, and alanine) were ubiquitously present, with average enantiomeric (D/L) ratios of 0.34, 0.26, 0.21, and 0.61 for Seoul, and 0.18, 0.11, 0.09, and 0.31 for Uljin, respectively. The much higher D/L ratios observed at Seoul than at Uljin might result from more advanced diagenetic stages as well as higher contributions from bacteria inhabiting terrestrial environments. The C- and N-normalized yields of D-alanine in DOM of our samples were found to be comparable to literature values reported for aquatic systems, where a significant portion of DOM was suggested to be of bacterial origin. Our study suggests that bacteria and their remnants might constitute an important fraction of OM in the atmosphere, contributing significantly to the quality of atmospheric OM and its post-depositional bioavailability in the surface ecosystems.

  4. Evaluation of Galactose Adapted Yeasts for Bioethanol Fermentation from Kappaphycus alvarezii Hydrolyzates.

    Science.gov (United States)

    Nguyen, Trung Hau; Ra, Chae Hun; Sunwoo, In Yung; Jeong, Gwi-Taek; Kim, Sung-Koo

    2016-07-28

    Bioethanol was produced from Kappaphycus alvarezii seaweed biomass using separate hydrolysis and fermentation (SHF). Pretreatment was evaluated for 60 min at 121°C using 12% (w/v) biomass slurry with 364 mM H2SO4. Enzymatic saccharification was then carried out at 45°C for 48 h using Celluclast 1.5 L. Ethanol fermentation with 12% (w/v) K. alvarezii hydrolyzate was performed using the yeasts Saccharomyces cerevisiae KCTC1126, Kluyveromyces marxianus KCTC7150, and Candida lusitaniae ATCC42720 with or without prior adaptation to high concentrations of galactose. When non-adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 11.5 g/l, 6.7 g/l, and 6.0 g/l of ethanol were produced, respectively. When adapted S. cerevisiae, K. marxianus, and C. lusitaniae were used, 15.8 g/l, 11.6 g/l, and 13.4 g/l of ethanol were obtained, respectively. The highest ethanol concentration was 15.8 g/l, with YEtOH = 0.43 and YT% = 84.3%, which was obtained using adapted S. cerevisiae.

  5. Batch dark fermentation from enzymatic hydrolyzed food waste for hydrogen production.

    Science.gov (United States)

    Han, Wei; Ye, Min; Zhu, Ai Jun; Zhao, Hong Ting; Li, Yong Feng

    2015-09-01

    A combination bioprocess of solid-state fermentation (SSF) and dark fermentative hydrogen production from food waste was developed. Aspergillus awamori and Aspergillus oryzae were utilized in SSF from food waste to generate glucoamylase and protease which were used to hydrolyze the food waste suspension to get the nutrients-rich (glucose and free amino nitrogen (FAN)) hydrolysate. Both glucose and FAN increased with increasing of food waste mass ratio from 4% to 10% (w/v) and the highest glucose (36.9 g/L) and FAN (361.3mg/L) were observed at food waste mass ratio of 10%. The food waste hydrolysates were then used as the feedstock for dark fermentative hydrogen production by heat pretreated sludge. The best hydrogen yield of 39.14 ml H2/g food waste (219.91 ml H2/VSadded) was achieved at food waste mass ratio of 4%. The proposed combination bioprocess could effectively accelerate the hydrolysis rate, improve raw material utilization and enhance hydrogen yield. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Effect of partially hydrolyzed guar gum (PHGG) on the bioaccessibility of fat and cholesterol.

    Science.gov (United States)

    Minekus, Mans; Jelier, Mark; Xiao, Jin-Zhong; Kondo, Shizuki; Iwatsuki, Keiji; Kokubo, Sadayuki; Bos, Martin; Dunnewind, Bertus; Havenaar, Robert

    2005-05-01

    The addition of a compound that lowers the intestinal uptake of fat and cholesterol might be an interesting strategy to reduce the risk of vascular disease. Partially hydrolyzed guar gum (PHGG) has been shown to have this effect in healthy volunteers after intake of a yogurt drink with 3 to 6% PHGG. In the present study a yogurt drink with 3% sunflower oil and 4% egg yolk was tested with 3% and 6% PHGG, and compared to a control without PHGG. Experiments were performed in a multi-compartmental model of the gastrointestinal tract, equipped to study the digestion and availability for absorption (bioaccessibility) of lipids. The results show that PHGG decreases the bioaccessibility of both fat and cholesterol in a dose-dependent manner. The bioaccessibility of fat was 79.4+/-1.7%, 70.8+/-2.5% and 60.1+/-1.1% for the control experiments and the experiments with 3% and 6% PHGG respectively. The bioaccessibility of cholesterol was 82.2+/-2.0%, 75.4+/-1.2% and 64.0+/-4.3% for the control and the experiments with 3% and 6% PHGG respectively. Additional experiments indicated that PHGG reduces bioaccessibility through the depletion flocculation mechanism. Depletion flocculation antagonizes the emulsification by bile salts and thus decreases lipolytic activity, resulting in a lower bioaccessibility of fat and cholesterol. Depletion flocculation with polymers might be an interesting mechanism, not described before, to reduce fat and cholesterol absorption.

  7. Identification of a Saccharomyces cerevisiae Glucosidase That Hydrolyzes Flavonoid Glucosides▿ †

    Science.gov (United States)

    Schmidt, Sabine; Rainieri, Sandra; Witte, Simone; Matern, Ulrich; Martens, Stefan

    2011-01-01

    Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-β-glucoside revealed considerable β-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic α- or β-glucosides identified three efficient β-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid β-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7- or 4′-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides. PMID:21216897

  8. Hydrolyzed guar gum decreases postprandial blood glucose and glucose absorption in the rat small intestine.

    Science.gov (United States)

    Takahashi, Toru; Yokawa, Takeo; Ishihara, Noriyuki; Okubo, Tsutomu; Chu, Djong-Chi; Nishigaki, Eri; Kawada, Yuka; Kato, Masako; Raj Juneja, Lekh

    2009-06-01

    We hypothesized that infusing partially hydrolyzed guar gum (PHGG) into the duodenum would reduce increases in postprandial plasma glucose by decreasing the rate of glucose diffusion from the small intestine luminal digesta of the rat. The postprandial plasma glucose and apparent glucose disappearance from the small intestine were measured after infusing artificial digesta containing 0 (control), 3.0, or 6.0 g/L PHGG into the duodenum via a cannula under anesthesia in experiments 1 and 2. The diffusion of glucose in the artificial digesta was estimated using dialysis tubing, filled with the same artificial digesta, soaked in a buffer in experiment 3. In experiment 1, the plasma glucose concentration was lower in the digesta containing 3.0 and 6.0 g/L PHGG than in the control digesta at 120 minutes (P glucose and insulin (experiment 1), apparent disappearance of glucose in the lumen of the small intestine (experiment 2), and net disappearance of glucose in the dialysis tube depended negatively on the viscosity of the artificial digesta (P postprandial blood glucose by lowering the rate of absorption from the small intestine in the rat by reducing the diffusion of glucose in the lumen.

  9. Oral Immunotherapy With Partially Hydrolyzed Wheat-Based Cereals: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Roger Lauener

    2017-09-01

    Full Text Available To date, only few studies have assessed oral immunotherapy (OIT for wheat allergy and often describe severe adverse reactions during therapy. We developed partially hydrolyzed wheat-based cereals (pHC, which were used in a multicenter, open-label, OIT pilot study, in immunoglobulin E–mediated wheat allergy children (NCT01332084. The primary objective of the study was to test whether wheat allergic patients tolerate pHC and primary end point was the presence or not of immediate adverse reactions to pHC during the 1-day initial escalation phase (stepwise increased doses of pHC, with evaluation of the maximum dose tolerated. Of the 9 patients enrolled in the trial, 4 discontinued OIT because of mild to severe reactions at the initial escalation phase. The 5 patients who passed the escalation phase consumed pHC daily for 1 to 6 months. One of these patients withdrew due to noncompliance, whereas the 4 others completed the study and successfully passed the wheat challenge test at the end of the study. About 60% of the adverse events were unrelated to the study product. Our study provides preliminary evidence that pHC is tolerated by a subset of wheat allergic patients. Further studies are warranted to test its efficacy as a potential therapeutic option for wheat allergic patients.

  10. Potentially bioavailable natural organic carbon and hydrolyzable amino acids in aquifer sediments

    Science.gov (United States)

    Thomas, Lashun K.; Widdowson, Mark A.; Novak, John T.; Chapelle, Francis H.; Benner, Ronald; Kaiser, Karl

    2012-01-01

    This study evaluated the relationship between concentrations of operationally defined potentially bioavailable organic -carbon (PBOC) and hydrolyzable amino acids (HAAs) in sediments collected from a diverse range of chloroethene--contaminated sites. Concentrations of PBOC and HAA were measured using aquifer sediment samples collected at six selected study sites. Average concentrations of total HAA and PBOC ranged from 1.96 ± 1.53 to 20.1 ± 25.6 mg/kg and 4.72 ± 0.72 to 443 ± 65.4 mg/kg, respectively. Results demonstrated a statistically significant positive relationship between concentrations of PBOC and total HAA present in the aquifer sediment (p amino acids are known to be readily biodegradable carbon compounds, this relationship suggests that the sequential chemical extraction procedure used to measure PBOC is a useful indicator of bioavailable carbon in aquifer sediments. This, in turn, is consistent with the interpretation that PBOC measurements can be used for estimating the amount of natural organic carbon available for driving the reductive dechlorination of chloroethenes in groundwater systems.

  11. Rheology of diluted and semi-diluted partially hydrolyzed polyacrylamide solutions under shear: Experimental studies

    Directory of Open Access Journals (Sweden)

    Rui Zhang

    2017-06-01

    Full Text Available Rheological properties of hydrolyzed polyacrylamide (HPAM solutions were measured in oscillatory and flow shear. In oscillatory shear the storage and loss moduli increased as the concentrations of the solutions increased, but they decreased as salinity increased. The relaxation spectra were plotted using the Kontogiorgos method. As shown in the relaxation spectra, the number of density of segmental units increased at first and then the peaks shifted and split into several as the concentration increased. With salinity increasing, the number of motion units increase and the peaks of segmental units became broader. In flow shear tests, the curves of viscosity versus shear rate were fitted by a power law equation. The result showed that the flow behavior index decreased with increasing concentration, while it generally increased with increasing of salinity. Furthermore, the first normal stress difference increased as the concentration was increased, while it decreased as the salinity was increased. The first normal stress differences measured by the rheometer were compared with the first normal stress differences calculated by the Laun equation and the results showed that Laun equation deviates the experimental results.

  12. Kinetics and thermodynamics of biodegradation of hydrolyzed polyacrylamide under anaerobic and aerobic conditions.

    Science.gov (United States)

    Zhao, Lanmei; Bao, Mutai; Yan, Miao; Lu, Jinren

    2016-09-01

    Kinetics and thermodynamics of hydrolyzed polyacrylamide (HPAM) biodegradation in anaerobic and aerobic activated sludge biochemical treatment systems were explored to determine the maximum rate and feasibility of HPAM biodegradation. The optimal nutrient proportions for HPAM biodegradation were determined to be 0.08g·L(-1) C6H12O6, 1.00g·L(-1) NH4Cl, 0.36g·L(-1) NaH2PO4 and 3.00g·L(-1) K2HPO4 using response surface methodology (RSM). Based on the kinetics, the maximum HPAM biodegradation rates were 16.43385mg·L(-1)·d(-1) and 2.463mg·L(-1)·d(-1) in aerobic and anaerobic conditions, respectively. The activation energy (Ea) of the aerobic biodegradation was 48.9897kJ·mol(-1). Entropy changes (ΔS) of biochemical treatment system decreased from 216.21J·K(-1) to 2.39J·K(-1). Thermodynamic windows of opportunity for HPAM biodegradation were drawn. And it demonstrated HPAM was biodegraded into acetic acid and CO2 under laboratory conditions. Growth-process equations for functional bacteria anaerobically grown on polyacrylic acid were constructed and it confirmed electron equivalence between substrate and product. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Acute simvastatin inhibits K ATP channels of porcine coronary artery myocytes.

    Directory of Open Access Journals (Sweden)

    Sai Wang Seto

    Full Text Available BACKGROUND: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular KATP channel gatings are unknown. METHODS: Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca(2+]i, [ATP]i and [glucose]o uptake measurements. RESULTS: The cromakalim (10 nM to 10 µM- and pinacidil (10 nM to 10 µM-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM. Simvastatin (1, 3 and 10 µM suppressed (in okadaic acid (10 nM-sensitive manner cromakalim (10 µM- and pinacidil (10 µM-mediated opening of whole-cell KATP channels of arterial myocytes. Simvastatin (10 µM and AICAR (1 mM elicited a time-dependent, compound C (1 µM-sensitive [(3H]-2-deoxy-glucose uptake and an increase in [ATP]i levels. A time (2-30 min- and concentration (0.1-10 µM-dependent increase by simvastatin of p-AMPKα-Thr(172 and p-PP2A-Tyr(307 expression was observed. The enhanced p-AMPKα-Thr(172 expression was inhibited by compound C, ryanodine (100 µM and KN93 (10 µM. Simvastatin-induced p-PP2A-Tyr(307 expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM, ouabain (10 µM, and in [glucose]o-free or [Na(+]o-free conditions. CONCLUSIONS: Simvastatin causes ryanodine-sensitive Ca(2+ release which is important for AMPKα-Thr(172 phosphorylation via Ca(2+/CaMK II. AMPKα-Thr(172 phosphorylation causes [glucose]o uptake (and an [ATP]i increase, closure of KATP channels, and phosphorylation of AMPKα-Thr(172 and PP2A-Tyr(307 resulted. Phosphorylation of PP2A-Tyr(307 occurs at a site downstream of AMPKα-Thr(172 phosphorylation.

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

    Directory of Open Access Journals (Sweden)

    Marie Elizabeth Gibbs

    2016-01-01

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

  15. 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

    ATP-sensitive potassium (K(ATP)) channels play an important role in the regulation of cerebral vascular tone. In vitro studies using synthetic K(ATP) channel openers suggest that the pharmacological profiles differ between rat basilar arteries and rat middle cerebral arteries. To address this issue......, 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...

  16. In vitro Scleroderma laeve and Eucalyptus grandis mycorrhization and analysis of atp6, 17S rDNA, and ras gene expression during ectomycorrhizal formation.

    Science.gov (United States)

    de Freitas Pereira, Maíra; Betancourth, Blanca Mercedes Leguízamo; Teixeira, Janaina Aparecida; Zubieta, Mariane Paludetti; de Queiroz, Marisa Vieira; Kasuya, Maria Catarina Megumi; Costa, Mauricio Dutra; de Araújo, Elza Fernandes

    2014-12-01

    The interaction between fungi and plants that form ectomycorrhizae (ECM) promotes alterations in the gene expression profiles of both organisms. Fungal genes expression related to metabolism were evaluated at the pre-symbiotic stage and during the ECM development between Scleroderma laeve and Eucalyptus grandis. Partial sequences of ATP synthase (atp6), translation elongation factor (ef1α), the RAS protein (ras), and the 17S rDNA genes were isolated. The expression of the atp6 and 17S rDNA genes during the pre-symbiotic stage showed an approximately threefold increase compared to the control. During ECM development, the expression of the 17S rDNA gene showed a 4.4-fold increase after 3 days of contact, while the expression of the atp6 gene increased 7.23-fold by the 15th day, suggesting that protein synthesis and respiratory chain activities are increased during the formation of the mantle and the Hartig net. The ras gene transcripts were only detected by RT-PCR 30 days after fungus-plant contact, suggesting that RAS-mediated signal transduction pathways are functional during the establishment of symbiosis. The present study demonstrates that alterations in gene expression occur in response to stimuli released by the plant during ECM association and increases the understanding of the association between S. laeve and E. grandis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. 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.

  18. E-NTPDase1/CD39 modulates renin release from heart mast cells during ischemia/reperfusion: a novel cardioprotective role.

    Science.gov (United States)

    Aldi, Silvia; Marino, Alice; Tomita, Kengo; Corti, Federico; Anand, Ranjini; Olson, Kim E; Marcus, Aaron J; Levi, Roberto

    2015-01-01

    Ischemia/reperfusion (I/R) elicits renin release from cardiac mast cells (MC), thus activating a local renin-angiotensin system (RAS), culminating in ventricular fibrillation. We hypothesized that in I/R, neurogenic ATP could degranulate juxtaposed MC and that ecto-nucleoside triphosphate diphosphohydrolase 1/CD39 (CD39) on MC membrane could modulate ATP-induced renin release. We report that pharmacological inhibition of CD39 in a cultured human mastocytoma cell line (HMC-1) and murine bone marrow-derived MC with ARL67156 (100 µM) increased ATP-induced renin release (≥2-fold), whereas purinergic P2X7 receptors (P2X7R) blockade with A740003 (3 µM) prevented it. Likewise, CD39 RNA silencing in HMC-1 increased ATP-induced renin release (≥2-fold), whereas CD39 overexpression prevented it. Acetaldehyde, an I/R product (300 µM), elicited an 80% increase in ATP release from HMC-1, in turn, causing an autocrine 20% increase in renin release. This effect was inhibited or potentiated when CD39 was overexpressed or silenced, respectively. Moreover, P2X7R silencing prevented ATP- and acetaldehyde-induced renin release. I/R-induced RAS activation in ex vivo murine hearts, characterized by renin and norepinephrine overflow and ventricular fibrillation, was potentiated (∼2-fold) by CD39 inhibition, an effect prevented by P2X7R blockade. Our data indicate that by regulating ATP availability at the MC surface, CD39 modulates local renin release and thus, RAS activation, ultimately exerting a cardioprotective effect. © FASEB.

  19. News/Press Releases

    Data.gov (United States)

    Office of Personnel Management — A press release, news release, media release, press statement is written communication directed at members of the news media for the purpose of announcing programs...

  20. A computational search for lipases that can preferentially hydrolyze long-chain omega-3 fatty acids from fish oil triacylglycerols.

    Science.gov (United States)

    Kamal, Md Zahid; Barrow, Colin J; Rao, Nalam Madhusudhana

    2015-04-15

    Consumption of long-chain omega-3 fatty acids is known to decrease the risk of major cardiovascular events. Lipases, a class of triacylglycerol hydrolases, have been extensively tested to concentrate omega-3 fatty acids from fish oils, under mild enzymatic conditions. However, no lipases with preference for omega-3 fatty acids selectivity have yet been discovered or developed. In this study we performed an exhaustive computational study of substrate-lipase interactions by docking, both covalent and non-covalent, for 38 lipases with a large number of structured triacylglycerols containing omega-3 fatty acids. We identified some lipases that have potential to preferentially hydrolyze omega-3 fatty acids from structured triacylglycerols. However omega-3 fatty acid preferences were found to be modest. Our study provides an explanation for absence of reports of lipases with omega-3 fatty acid hydrolyzing ability and suggests methods for developing these selective lipases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

    DEFF Research Database (Denmark)

    Beuschlein, Felix; Boulkroun, Sheerazed; Osswald, Andrea

    2013-01-01

    Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α...... subunit) and ATP2B3 (encoding a Ca2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced...... affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation...

  2. Hydrolyzable hemoglobin adducts of polyfunctional monocyclic N-substituted arenes as dosimeters of exposure and markers of metabolism.

    Science.gov (United States)

    Zwirner-Baier, I; Kordowich, F J; Neumann, H G

    1994-01-01

    Hemoglobin adducts of 10 polyfunctional amino- and nitro-substituted benzenes and toluenes were analyzed: 2,4,6-trinitrotoluene, 2,4- and 2,6-dinitrotoluene, 2-amino-4-nitrotoluene, 4-amino-2-nitrotoluene, 2,4- and 2,6-diaminotoluene, 1,3-dinitrobenzene, 1-amino-3-nitrobenzene, and 1,3-diaminobenzene. A single dose (0.5 mmole/kg) of the test compounds was administered to female Wistar rats by gavage, and blood extracted and hemoglobin prepared after 24 hr. One or more cleavage products could be obtained in each case by hydrolyzing hemoglobin (Hb). Hemoglobin binding indices (HBI: binding [mmole/mole Hb]/dose [mmole/kg]) and the ratios of hydrolyzable adducts were determined. The HBI ranged between < 0.02 and 69.0. The results indicate the in vivo formation of several, covalently bound, hydrolyzable hemoglobin adducts. Conclusions on prevailing metabolic pathways can be drawn. Total binding of several compounds seems sufficient for biomonitoring of human blood samples. These chemicals are considered representative for environmental contamination with explosives of this type, and we propose their Hb adducts be used as dosimeters for human exposure to these suspected carcinogens. PMID:7889857

  3. Experimental and theoretical study of the influence of water on hydrolyzed product formation during the feruloylation of vegetable oil.

    Science.gov (United States)

    Compton, David L; Evans, Kervin O; Appell, Michael

    2017-07-01

    Feruloylated vegetable oil is a valuable green bioproduct that has several cosmeceutical applications associated with its inherent anti-oxidant and ultraviolet-absorption properties. Hydrolyzed vegetable oil by-products can influence product quality and consistency. The formation of by-products by residual water in the enzymatic synthesis of feruloylated vegetable oil was investigated using chemical theory and experimental studies by monitoring the reaction over a 22-day period. The hydrolysis of vegetable oil is thermodynamically favored over the hydrolysis of the ethyl ferulate starting material. These results suggest that hydrolyzed vegetable oil products will be experimentally observed in greater concentrations compared to hydrolyzed ethyl ferulate products. Quantum chemical studies identified several reaction mechanisms that explain the formation of side products by water, suggesting that residual water influences product quality. Efforts to reduce residual water can improve product consistency and reduce purification costs. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  4. Purification and Characterization of a Ginsenoside Rb1-Hydrolyzing β-Glucosidase from Aspergillus niger KCCM 11239

    Directory of Open Access Journals (Sweden)

    Kyung Hoon Chang

    2012-09-01

    Full Text Available Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu2+, Mg2+, Co2+, and acetic acid (10 mM. In the specificity tests, the enzyme was found to be active against ginsenoside Rb1, but showed very low levels of activity against Rb2, Rc, Rd, Re, and Rg1. The enzyme hydrolyzed the 20-C,β-(1→6-glucoside of ginsenoside Rb1 to generate ginsenoside Rd and Rg3, and hydrolyzed 3-C,β-(1→2-glucoside to generate F2. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds.

  5. Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA.

    Science.gov (United States)

    Liu, Chunlan; Cai, Dawei; Zhang, Lin; Tang, Wei; Yan, Ran; Guo, Haitao; Chen, Xulin

    2016-10-01

    The development of new agents to target HBV cccDNA is urgently needed because of the limitations of current available drugs for treatment of hepatitis B. By using a cell-based assay in which the production of HBeAg is in a cccDNA-dependent manner, we screened a compound library derived from Chinese herbal remedies for inhibitors against HBV cccDNA. Three hydrolyzable tannins, specifically punicalagin, punicalin and geraniin, emerged as novel anti-HBV agents. These compounds significantly reduced the production of secreted HBeAg and cccDNA in a dose-dependent manner in our assay, without dramatic alteration of viral DNA replication. Furthermore, punicalagin did not affect precore/core promoter activity, pgRNA transcription, core protein expression, or HBsAg secretion. By employing the cell-based cccDNA accumulation and stability assay, we found that these tannins significantly inhibited the establishment of cccDNA and modestly facilitated the degradation of preexisting cccDNA. Collectively, our results suggest that hydrolyzable tannins inhibit HBV cccDNA production via a dual mechanism through preventing the formation of cccDNA and promoting cccDNA decay, although the latter effect is rather minor. These hydrolyzable tannins may serve as lead compounds for the development of new agents to cure HBV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Biohydrogen production from untreated and hydrolyzed potato steam peels by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

    Energy Technology Data Exchange (ETDEWEB)

    Mars, Astrid E.; Veuskens, Teun; Budde, Miriam A.W.; van Doeveren, Patrick F.N.M.; Lips, Steef J.; Bakker, Robert R.; de Vrije, Truus; Claassen, Pieternel A.M. [Wageningen UR, Food and Biobased Research, P.O. Box 17, 6700 AA Wageningen (Netherlands)

    2010-08-15

    Production of hydrogen by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana was studied in serum flasks and in pH-controlled bioreactors with glucose, and hydrolyzed and untreated potato steam peels (PSP) as carbon sources. Two types of PSP hydrolysates were used: one in which the starch in the PSP was liquefied with alpha-amylase, and one in which the liquefied starch was further hydrolyzed to glucose by amyloglucosidase. When the PSP hydrolysates or untreated PSP were added at circa 10-14 g/L of glucose units, both strains grew well and produced hydrogen with reasonable to high molar yields (2.4-3.8 moles H{sub 2}/mole glucose units), and no significant production of lactate. The hydrogen production rates and yields were similar with untreated PSP, hydrolyzed PSP, and pure glucose, showing that C. saccharolyticus and T. neapolitana are well equipped for the utilization of starch. When the concentrations of the substrates were increased, growth and hydrogen production of both strains were hampered. At substrate concentrations of circa 30-40 g/L of glucose units, the molar hydrogen yield of C. saccharolyticus was severely reduced due to the formation of high amounts of lactate, while T. neapolitana was unable to grow at all. The results showed that PSP and PSP hydrolysates are very suitable substrates for efficient fermentative hydrogen production at moderate substrate loadings. (author)

  7. [Effect of extensively hydrolyzed formula on growth and development of infants with very/extremely low birth weight].

    Science.gov (United States)

    Gu, Chun-Yan; Jiang, Hui-Fen; Wang, Jin-Xiu

    2017-08-01

    To study the effect of extensively hydrolyzed formula on the growth and development in very low birth weight (VLBW) and extremely low birth weight (ELBW) infants. A total of 375 VLBW or ELBW infants were enrolled and divided into an observation group (187 infants) and a control group (188 infants) using a random number table. The infants in the observation group were given extensively hydrolyzed formula, and when the amount of extensively hydrolyzed formula reached 10 mL/time, it was changed to the standard formula for preterm infants. The infants in the control group were given standard formula for preterm infants. Both groups were fed for 4 consecutive weeks and were compared in terms of incidence rate of feeding intolerance, time to establish full enteral feeding, time to complete meconium excretion, number of spontaneous bowel movements, growth and development, motilin level at 4 and 10 days after feeding, and incidence rate of infection. Compared with the control group, the observation group had a lower rate of feeding intolerance (Pgrowth and development, and reduce the incidence of infection in VLBW and ELBW infants.

  8. 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.

  9. Release of bisphenol A from polycarbonate: a review.

    Science.gov (United States)

    Hoekstra, Eddo J; Simoneau, Catherine

    2013-01-01

    The release of Bisphenol A (BPA) from polycarbonate baby bottles into food and food simulants is reviewed in the perspective of the current intensive discussions on the risks of this substance. Potential factors that have been reported to influence the release of BPA are reviewed. Unlike most polymers polycarbonate is hydrolyzed under alkaline conditions by scale formation, residual alkaline detergents and boiled water. Data suggest that brushing of the bottle did not raise the release of BPA. Claims that used bottles release more BPA than new bottles and that mineral composition of the aqueous food simulant affect release could not be substantiated. There are indications that aminolysis of polycarbonate by milk and ethanolysis of polycarbonate by 50% ethanol might take place under relevant test conditions. The relatively few migration data following the test conditions of European food contact material legislation, comply with the specific migration limit. Two test conditions were identified that reflect real use and exposure, and might cause higher release of BPA compared to the test conditions of European food contact material legislation. Further detailed studies are necessary to verify whether these two exposure scenarios are more severe.

  10. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...

  11. Hypotonic shock stimulates ascorbate release from coronary artery endothelial cells by a Ca2+ -independent pathway.

    Science.gov (United States)

    Davis, Kim A; Samson, Sue E; Wilson, John X; Grover, Ashok K

    2006-10-24

    In endothelial cells, anion channels open upon osmotic swelling during shear stress and hypotonic shock. Therefore, we examined the effects of hypotonic shock on release of the antioxidant anion ascorbate from pig coronary artery endothelial cells. Hypotonic shock potentiated ascorbate release from freshly isolated or cultured pig coronary artery endothelial cells; subsequently cultured endothelial cells were used. The hypotonic shock-induced increase in Asc release was rapid, depended on the degree of hypotonic shock, and not due to membrane leakiness. Stimulating P2Y2 like receptors in endothelial cells with ATP causes ascorbate release via a Ca2+ -mediated pathway. Hypotonic shock-induced release differed from the Ca2+-mediated Asc release because: (a) the increase in release with hypotonic shock was additive to that with ATP or A23187 (Ca2+ -ionophore), (b) apyrase, suramin or removing extracellular Ca2+ did not affect the hypotonic shock-stimulated release, (c) anion channel blockers inhibited the release by the two pathways differently, and (d) hypotonic shock increased the ascorbate release from endothelial cells and cultured smooth muscle cells whereas the Ca2+ -mediated ascorbate release occurred only in endothelial cells. Accumulation of ascorbate by endothelial cells was examined at extracellular ascorbate concentrations of 10 (Na+ -ascorbate symporter not saturated) and 5000 microM (Na+ -ascorbate symporter saturated). Hypotonic shock and A23187 decreased ascorbate accumulation at 10 microM ascorbate but increased it at 5000 microM. The effects of the two treatments were additive and also differed from each other with substitution of gluconate for extracellular chloride. Thus, ascorbate release from endothelial cells can be potentiated by two distinct pathways - hypotonic shock mediated and ATP/Ca2+ stimulated.

  12. Epidemiological link between wheat allergy and exposure to hydrolyzed wheat protein in facial soap.

    Science.gov (United States)

    Fukutomi, Y; Taniguchi, M; Nakamura, H; Akiyama, K

    2014-10-01

    Recent studies have highlighted the importance of extra-intestinal routes of sensitization to food-related allergens as the cause of epidemics of food allergy. Instances of Japanese women developing food allergy to wheat after exposure to hydrolyzed wheat protein (HWP) present in facial soap have been reported. However, the epidemiologic impact of these ingredients as a cause of food allergy has not been well studied. To clarify the epidemiological relationship between food allergy to wheat and contact exposure to HWP, a case-control study of Japanese women aged 20-54 years with self-reported wheat allergy (WA) (cases, n = 157) and age-matched control subjects without WA (controls, n = 449) was performed using a large-scale Web-based research panel. Subjects answered a Web-based questionnaire regarding the use of skin and hair care products, as well as other possible risk factors. Current use of an HWP-containing facial soap (Cha no Shizuku; Yuka) was significantly associated with an increased risk of WA (adjusted odds ratio, 2.6; 95% confidence interval, 1.2-5.7; frequencies of current use in cases and controls; 11% and 6%, respectively). Use of Cha no Shizuku was more common in subjects with more recent-onset WA, implying that this soap may have contributed to the recent epidemic of WA. An epidemiological relationship between WA and contact exposure to HWP has been documented. This study implicates a possible role of contact exposure to food-derived protein hydrolysates as a risk factor for the development of food allergy manifesting itself as anaphylaxis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Different composition and distribution patterns of mineral-protected versus hydrolyzable lipids in shrubland soils

    Science.gov (United States)

    Cai, Yue; Tang, Zhiyao; Xiong, Gaoming; Xie, Zongqiang; Liu, Zongguang; Feng, Xiaojuan

    2017-09-01

    Mineral protection is known as an important mechanism stabilizing soil organic carbon (SOC). However, the composition, sources, and variations of mineral-protected SOC remain poorly constrained. To fill this knowledge gap, we used hydrofluoric acid to demineralize soil matrix and compared the sources and distribution of mineral-protected lipids (ML) versus hydrolyzable lipids (HL) of four typical Chinese shrubland soils. ML was found to represent a sizable fraction (9-32%) of total aliphatic lipids (including n-alkanols; n-alkanoic acids; α,ω-alkanedioic acids; hydroxyalkanoic acids; and midchain-substituted acids) in all soils. Based on carbon chain length and branch positions, microbe- and plant-derived lipids were distinguished. No significant difference was found in the ratio of microbe- to plant-derived lipids in ML versus HL, implying that plant and microbial inputs are equally important for the mineral-associated soil lipids. However, ML contained a higher proportion of nonspecific lipids, especially at depths. Furthermore, to evaluate key environmental variable(s) controlling the distribution of different lipid components, a multiple stepwise regression analysis was conducted. Notably, ML was mainly affected by SOC-to-nitrogen ratio instead of mineralogical properties, implying that the accrual of mineral-associated soil lipids relies strongly on organic matter properties. Collectively, our findings provide novel insights on sources and accumulation mechanisms of mineral-protected soil lipids. SOC decomposition and subsequent accretion of degradation products appear to be vital for the sequestration of mineral-associated soil lipids and warrant better recognition in the investigations of stable soil carbon accumulation mechanisms.

  14. Anti-inflammatory and analgesic potential of hydrolyzed extract of Agave sisalana Perrine ex Engelm., Asparagaceae

    Directory of Open Access Journals (Sweden)

    Ricardo J. Dunder

    Full Text Available The hemolytic, anti-inflammatory and antinociceptive properties from hydrolyzed extract Agave sisalana Perrine ex Engelm., Asparagaceae (HEAS was evaluated on classic inflammation models. Male Swiss mice and male Wistars rats received HEAS (500 mg/kg in two administration p.o. and i.p. in saline solution 0.9%. The acid hydrolysis inhibited the hemolytic action of saponins due to the retreat of side chain sugar. The treatment of the ear induced oedema by xylene with HEAS significantly reduced in two routes 13±1.5 and 10±0.63 mg, respectively, p.o. and i.p., in comparison with controls 27±1.5 saline and 13.5±1.2 AAS. The HEAS also diminished edema induced by carrageenin 43±1.58 mg (p.o. and 17±1.26 mg (i.p., when compared with control groups 52±1.58 mg (saline and 10.05±1.58 (indomethacin. HEAS showed analgesic effects in abdominal constrictions 30.7% (p.o., 88.7% (i.p. comparable to that produced by (AAS 70.6%. However in granuloma cotton pellet a chronic model of inflammation just the i.p. pathway decreased granulomatous tissue (20.4±1.32 mg compared with controls 30.5±2.53 mg (saline and 20.2±2.18 mg (dexamethasone. These data suggest that HEAS has anti-inflammatory and analgesic activity on acute and chronic processes.

  15. Hydrocarbon fuels from gas phase decarboxylation of hydrolyzed free fatty acid

    KAUST Repository

    Wang, Weicheng

    2012-01-01

    Gas phase decarboxylation of hydrolyzed free fatty acid (FFA) from canola oil has beeninvestigated in two fix-bed reactors by changing reaction parameters such as temperatures,FFA feed rates, and H 2-to-FFA molar ratios. FFA, which contains mostly C 18 aswell as a few C 16, C 20, C 22, and C 24 FFA, was fed into the boiling zone, evaporated, carriedby hydrogen flow at the rate of 0.5-20 ml/min, and reacted with the 5% Pd/C catalystin the reactor. Reactions were conducted atmospherically at 380-450 °C and the products,qualified and quantified through gas chromatography-flame ionization detector(GC-FID), showed mostly n-heptadecane and a few portion of n-C 15, n-C 19, n-C 21, n-C 23 as well as some cracking species. Results showed that FFA conversion increased withincreasing reaction temperatures but decreased with increasing FFA feed rates and H 2-to-FFA molar ratios. The reaction rates were found to decrease with higher temperatureand increase with higher H 2 flow rates. Highly selective heptadecane was achieved byapplying higher temperatures and higher H 2-to-FFA molar ratios. From the results, ascatalyst loading and FFA feed rate were fixed, an optimal reaction temperature of 415 °C as well as H 2-to-FFA molar ratio of 4.16 were presented. These results provided goodbasis for studying the kinetics of decarboxylation process. © 2012 American Society of Mechanical Engineers.

  16. Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Zincke, Diansy; Balasubramanian, Deepak; Silver, Lynn L; Mathee, Kalai

    2016-02-01

    Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC β-lactamase. A second, chromosomally encoded β-lactamase, PoxB, has previously been reported in P. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frame ampC, poxB, and oprD deletions, as well as complementation by expression under the control of an inducible promoter. While poxB deletions failed to alter β-lactam sensitivities, expression of poxB in ampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in an ampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding gene oprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing β-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-β-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity. In vitro susceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition, poxB was found to be cotranscribed with an upstream open reading frame, poxA, which itself was shown to encode a 32-kDa protein of yet unknown function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Inhibition of oxidant-induced biochemical changes of pork myofibrillar protein by hydrolyzed potato protein.

    Science.gov (United States)

    Wang, L L; Xiong, Y L

    2008-08-01

    The objective of the study was to investigate the role of hydrolyzed potato protein (HPP) in protecting myofibril protein isolate (MPI) from oxidative modification. MPI prepared from pork muscle was suspended (30 mg protein/mL) in 15 mM piperazine-N, N-bis(2-ethane sulfonic acid) buffer (pH 6.0) with 0, 0.3, 0.75, and 1.5 mg/mL of antioxidative HPP (1-h Alcalase hydrolysate). Oxidation was induced by incubating the protein suspensions at 4 degrees C for 24 h with (1) an iron-catalyzed oxidizing system (IOS: 0.01 mM FeCl3, 0.1 mM ascorbic acid, and 1.0 mM H202) and (2) a metmyoglobin-oxidizing system (MOS: 0.1 mM metmyoglobin and 0.1 mM H2O2). Changes in oxidized MPI were measured as thiobarbituric acid-reactive substances (TBARS), protein carbonylcontent, Ca- and K-ATPase activities, and ultraviolet (UV) spectra. Oxidation increased the production of TBARS and protein carbonyls by 2.9- and 0.24-fold in IOS and 5.6- and 2.2-fold in MOS, respectively. The 2 oxidizing systems altered the Ca- and K-ATPase activities and exposed hydrophobic groups buried in MPI. The presence of HPP reduced the extent of MPI oxidation in all physicochemical categories tested. Therefore, HPP may be used as a potential functional ingredient in meat products to enhance their oxidative stability.

  18. Safety and toxicological evaluation of a novel, fermented, peptide-enriched, hydrolyzed swine placenta extract powder.

    Science.gov (United States)

    Mitsui, Yukio; Bagchi, Manashi; Marone, Palma Ann; Moriyama, Hiroyoshi; Bagchi, Debasis

    2015-01-01

    Placenta is an important organ that connects the developing fetus to allow nutrient uptake, antibody provisions and gas exchange via the blood supply of the mother. We developed a novel, standardized, stable, water-soluble, peptide-enriched hydrolyzed, Horus fermented placenta powder (HFPEP) from healthy, pathogen-free, swine placenta. Earlier studies demonstrated that HFPEP significantly improves physical fatigue, hepatic functions and repair of muscle fibers. We examined the broad safety of HFPEP in various toxicology models in Good Laboratory Practices-approved laboratories. The acute oral toxicity study was conducted in female Sprague-Dawley rats, and the acute oral LD50 was found to be greater than 5000 mg/kg body weight. Ames' bacterial reverse mutation assay was conducted to determine the ability of HFPEP to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of a metabolic activation system (S9) at the doses of 50, 15, 4.5, 1.35 and 0.41 mg/ml. No mutagenic potential was observed. Mutagenic potential was also evaluated using in vivo micronucleus test, and no mutagenic potential of HFPEP was observed. Repeated dose 28-d oral toxicity study was performed in male and female rats with 14-d recovery period at the dose levels of 250, 500 or 1000 mg/kg. No abnormal clinical signs or toxicity were detected. No observed adverse effect level of HFPEP was found to be greater than 1000 mg/kg body weight. These studies affirm that HFPEP has broad spectrum safety for human consumption.

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

    Science.gov (United States)

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

    1992-01-01

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

  20. ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology.

    Science.gov (United States)

    De Col, Valentina; Fuchs, Philippe; Nietzel, Thomas; Elsässer, Marlene; Voon, Chia Pao; Candeo, Alessia; Seeliger, Ingo; Fricker, Mark D; Grefen, Christopher; Møller, Ian Max; Bassi, Andrea; Lim, Boon Leong; Zancani, Marco; Meyer, Andreas J; Costa, Alex; Wagner, Stephan; Schwarzländer, Markus

    2017-07-18

    Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

  1. Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner

    Science.gov (United States)

    Hironaka, Ippei; Iwase, Tadayuki; Sugimoto, Shinya; Okuda, Ken-ichi; Tajima, Akiko; Yanaga, Katsuhiko

    2013-01-01

    ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that Enterococcus gallinarum, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci, Enterococcus mundtii secreted the greatest amount of ATP (>2 μM/108 cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from E. mundtii. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase E. mundtii and Enterococcus faecalis cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function. PMID:23354720

  2. Silencing of atp6v1c1 prevents breast cancer growth and bone metastasis.

    Science.gov (United States)

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

    2013-01-01

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

  3. Activation of ATP/UTP-selective receptors increases blood flow and blunts sympathetic vasoconstriction in human skeletal muscle

    DEFF Research Database (Denmark)

    Yegutkin, G.G.; Gonzalez-Alonso, J.; Rosenmeier, Jaya Birgitte

    2008-01-01

    and sympatholytic effects of exogenous ATP in the skeletal muscle vasculature are largely mediated via ATP itself rather than its dephosphorylated metabolites, most likely via binding to endothelial ATP/UTP-selective P2Y(2) receptors. These data are consistent with a role of ATP in skeletal muscle hyperaemia...

  4. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    NARCIS (Netherlands)

    Tjaden, J.; Haferkamp, I.; Boxma, B.; Tielens, A.G.; Huynen, M.A.; Hackstein, J.H.P.

    2004-01-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and

  5. The relationship between energy metabolism and the action of inhibitors of histamine release

    DEFF Research Database (Denmark)

    Garland, L G; Johansen, Torben

    1977-01-01

    1 Dextran-induced release of histamine from rat mast cells was inhibited equally in complete and glucose-free Tyrode solution by doxantrazole (0.03-3 micronmol/l), theophylline (0.1-3 mmol/l) and dicumarol (0.01-10 micronmol/litre). 2 Doxantrazole (3 micronmol/l), theophylline (3 mmol....... 4 These results suggest that dicumarol, like doxantrazole and theophylline, inhibits histamine release without affecting mast cell energy metabolism. In contrast, papaverine probably inhibits release by depleting ATP that is required for exocytosis. 5 Inhibition of histamine release by dibutyryl...

  6. 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

  7. Molecular dynamics simulation studies of GLUT4: substrate-free and substrate-induced dynamics and ATP-mediated glucose transport inhibition.

    Science.gov (United States)

    Mohan, Suma; Sheena, Aswathy; Poulose, Ninu; Anilkumar, Gopalakrishnapillai

    2010-12-03

    Glucose transporter 4 (GLUT4) is an insulin facilitated glucose transporter that plays an important role in maintaining blood glucose homeostasis. GLUT4 is sequestered into intracellular vesicles in unstimulated cells and translocated to the plasma membrane by various stimuli. Understanding the structural details of GLUT4 will provide insights into the mechanism of glucose transport and its regulation. To date, a crystal structure for GLUT4 is not available. However, earlier work from our laboratory proposed a well validated homology model for GLUT4 based on the experimental data available on GLUT1 and the crystal structure data obtained from the glycerol 3-phosphate transporter. In the present study, the dynamic behavior of GLUT4 in a membrane environment was analyzed using three forms of GLUT4 (apo, substrate and ATP-substrate bound states). Apo form simulation analysis revealed an extracellular open conformation of GLUT4 in the membrane favoring easy exofacial binding of substrate. Simulation studies with the substrate bound form proposed a stable state of GLUT4 with glucose, which can be a substrate-occluded state of the transporter. Principal component analysis suggested a clockwise movement for the domains in the apo form, whereas ATP substrate-bound form induced an anti-clockwise rotation. Simulation studies suggested distinct conformational changes for the GLUT4 domains in the ATP substrate-bound form and favor a constricted behavior for the transport channel. Various inter-domain hydrogen bonds and switching of a salt-bridge network from E345-R350-E409 to E345-R169-E409 contributed to this ATP-mediated channel constriction favoring substrate occlusion and prevention of its release into cytoplasm. These data are consistent with the biochemical studies, suggesting an inhibitory role for ATP in GLUT-mediated glucose transport. In the absence of a crystal structure for any glucose transporter, this study provides mechanistic details of the conformational

  8. Intracellular ATP concentration contributes to the cytotoxic and cytoprotective effects of adenosine.

    Directory of Open Access Journals (Sweden)

    Shujue Li

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

  9. 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.

  10. 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).

  11. K-ATP channel expression and pharmacological in vivo and in vitro studies of the K-ATP channel blocker PNU-37883A in rat middle meningeal arteries

    DEFF Research Database (Denmark)

    Ploug, K.B.; Boni, L.J.; Baun, M.

    2008-01-01

    intracranial arteries, including the middle meningeal artery (MMA). We studied the K-ATP channel expression profile in rat MMA and examined the potential inhibitory effects of the K-ATP channel blocker PNU-37883A on K-ATP channel opener-induced relaxation of the rat MMA, using the three K-ATP channel openers...... levcromakalim, pinacidil and P-1075. Experimental approach: mRNA and protein expression of K-ATP channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K-ATP channel drugs on rat MMA were studied in the genuine...... closed cranial window model and in myograph baths, respectively. Key results: Expression studies indicate that inwardly rectifying K+ (Kir)6.1/sulphonylurea receptor (SUR) 2B is the major K-ATP channel complex in rat MMA. PNU-37883A (0.5 mg kg(-1)) significantly inhibited the in vivo dilatory effect...

  12. Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity.

    Science.gov (United States)

    Serra, Montserrat; Brazís, Pilar; Fondati, Alessandra; Puigdemont, Anna

    2006-11-01

    To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. 8 naïve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.

  13. Chemical release module facility

    Science.gov (United States)

    Reasoner, D. L.

    1980-01-01

    The chemical release module provides the capability to conduct: (1) thermite based metal vapor releases; (2) pressurized gas releases; (3) dispersed liquid releases; (4) shaped charge releases from ejected submodules; and (5) diagnostic measurements with pi supplied instruments. It also provides a basic R-F and electrical system for: (1) receiving and executing commands; (2) telemetering housekeeping data; (3) tracking; (4) monitoring housekeeping and control units; and (5) ultrasafe disarming and control monitoring.

  14. The cotton ATP synthase δ1 subunit is required to maintain a higher ATP/ADP ratio that facilitates rapid fibre cell elongation.

    Science.gov (United States)

    Pang, Y; Wang, H; Song, W-Q; Zhu, Y-X

    2010-11-01

    The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthase activity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors of ATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation. © 2010 German Botanical Society and The Royal Botanical Society of The Netherlands.

  15. Treatment with Oral ATP decreases alternating hemiplegia of childhood with de novo ATP1A3 Mutation.

    Science.gov (United States)

    Ju, Jun; Hirose, Shinichi; Shi, Xiu-Yu; Ishii, Atsushi; Hu, Lin-Yan; Zou, Li-Ping

    2016-05-04

    Alternating hemiplegia of childhood is an intractable neurological disorder characterized by recurrent episodes of alternating hemiplegia accompanied by other paroxysmal symptoms. Recent research has identified mutations in the ATP1A3 gene as the underlying cause. Adenosine-5'-triphosphate has a vasodilatory effect, can enhance muscle strength and physical performance, and was hypothesized to improve the symptoms of paroxysmal hemiplegia. A 7-year-old boy with alternating hemiplegia of childhood who was positive for a de novo ATP1A3 mutation was treated with adenosine- 5'- triphosphate supplementation orally as an innovative therapy for 2 years. Outcome was evaluated through the follow-up of improvement of hemiplegic episodes and psychomotor development. Side effects and safety were monitored in regularity. With the dosage of adenosine-5'-triphosphate administration increased, the patient showed significantly less frequency and shorter duration of hemiplegic episodes. Treatment with adenosine-5'-triphosphate was correlated with a marked amelioration of alternating hemiplegia of childhood episodes, and psychomotor development has improved. The maximum dose of oral administration of adenosine-5'-triphosphate reached 25 mg/kg per day. Adenosine-5'-triphosphate therapy was well tolerated without complaint of discomfort and side effects. The 2-year follow-up outcome of adenosine-5'-triphosphate therapy for alternating hemiplegia of childhood was successful.

  16. Improved cycling performance with ingestion of hydrolyzed marine protein depends on performance level

    Directory of Open Access Journals (Sweden)

    Vegge Geir

    2012-04-01

    Full Text Available Abstract Background The effect on performance of protein ingestion during or after exercise is not clear. This has largely been attributed to the utilization of different scientific protocols and the neglection of accounting for factors such as differences in physical and chemical properties of protein supplements and differences in athletic performance level. Methods We hypothesized that ingestion of unprocessed whey protein (15.3 g·h-1 together with carbohydrate (60 g·h-1, would provide no ergogenic effect on 5-min mean-power performance following 120 min cycling at 50% of maximal aerobic power (2.8 ± 0.2 W·kg-1, corresponding to 60 ± 4% of VO2max, compared to CHO alone (60 g·h-1. Conversely, we hypothesized that ingestion of the hydrolyzed marine protein supplement NutriPeptin™ (Np, 2.7 g·h-1, a processed protein supplement with potentially beneficial amino acid composition, together with a PROCHO beverage (12.4 g·h-1 and 60 g·h-1, respectively would provide an ergogenic effect on mean-power performance. We also hypothesized that the magnitude of the ergogenic effect of NpPROCHO would be dependent on athletic performance. As for the latter analysis, performance level was defined according to a performance factor, calculated from individual pre values of Wmax, VO2max and 5-min mean-power performance, wherein the performance of each subject was ranked relative to the superior cyclist whos performance was set to one. Twelve trained male cyclists (VO2max = 65 ± 4 ml·kg-1·min-1 participated in a randomized double-blinded cross-over study. Results and conclusions Overall, no differences were found in 5-min mean-power performance between either of the beverages (CHO 5.4 ± 0.5 W·kg-1; PROCHO 5.3 ± 0.5 W·kg-1; NpPROCHO 5.4 ± 0.3 W·kg-1 (P = 0.29. A negative correlation was found between NpPROCHO mean-power performance and athletic performance level (using CHO-performance as reference; Pearson R = -0.74, P = 0.006. Moreover

  17. ATP binding by NLRP7 is required for inflammasome activation in response to bacterial lipopeptides.

    Science.gov (United States)

    Radian, Alexander D; Khare, Sonal; Chu, Lan H; Dorfleutner, Andrea; Stehlik, Christian

    2015-10-01

    Nucleotide-binding oligimerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) involved in innate immune responses. NLRs encode a central nucleotide-binding domain (NBD) consisting of the NAIP, CIITA, HET-E and TP1 (NACHT) domain and the NACHT associated domain (NAD), which facilitates receptor oligomerization and downstream inflammasome signaling. The NBD contains highly conserved regions, known as Walker motifs, that are required for nucleotide binding and hydrolysis. The NLR containing a PYRIN domain (PYD) 7 (NLRP7) has been recently shown to assemble an ASC and caspase-1-containing high molecular weight inflammasome complex in response to microbial acylated lipopeptides and Staphylococcus aureus infection. However, the molecular mechanism responsible for NLRP7 inflammasome activation is still elusive. Here we demonstrate that the NBD of NLRP7 is an ATP binding domain and has ATPase activity. We further show that an intact nucleotide-binding Walker A motif is required for NBD-mediated nucleotide binding and hydrolysis, oligomerization, and NLRP7 inflammasome formation and activity. Accordingly, THP-1 cells expressing a mutated Walker A motif display defective NLRP7 inflammasome activation, interleukin (IL)-1β release and pyroptosis in response to acylated lipopeptides and S. aureus infection. Taken together, our results provide novel insights into the mechanism of NLRP7 inflammasome assembly. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Cost-effectiveness of using an extensively hydrolyzed casein formula plus the probiotic Lactobacillus rhamnosus GG compared to an extensively hydrolyzed formula alone or an amino acid formula as first-line dietary management for cow’s milk allergy in the US

    Directory of Open Access Journals (Sweden)

    Ovcinnikova O

    2015-02-01

    management with eHCF + LGG appears to afford a more cost-effective use of health care resources than initial dietary management with eHCF or AAF since it releases health care resources for alternative use within the system and reduces costs without impacting on the time needed to manage the allergy. Keywords: amino acid formula, cost-effectiveness, cow’s milk allergy, economic evaluation, extensively hydrolyzed formula, Lactobacillus rhamnosus GG, Neocate, Nutramigen, US

  19. Quebrachitol-induced gastroprotection against acute gastric lesions: role of prostaglandins, nitric oxide and K+ ATP channels.

    Science.gov (United States)

    de Olinda, T M; Lemos, T L G; Machado, L L; Rao, V S; Santos, F A

    2008-05-01

    The effect of Quebrachitol (2-O-methyl-L-inositol), a bioactive component from Magonia glabrata fruit extract was investigated against gastric damage induced by absolute ethanol (96%, 0.2 ml/animal) and indomethacin (30 mg/kg, p.o.), in mice. Quebrachitol at oral doses of 12.5, 25, and 50mg/kg markedly attenuated the gastric lesions induced by ethanol to the extent of 69%, 64%, and 53% and against indomethacin by 55%, 59%, and 26%, respectively. While pretreatment with TRPV1 antagonist capsazepine (5mg/kg, i.p.) failed to block effectively the gastroprotective effect of quebrachitol (25mg/kg) against ethanol damage, the non-selective cyclooxygenase inhibitor indomethacin (10mg/kg, p.o.), almost abolished it. Furthermore, quebrachitol effect was significantly reduced in mice pretreated with L-NAME, or glibenclamide, the respective inhibitors of nitric oxide synthase and K(+)(ATP) channel activation. Thus we provide the first evidence that quebrachitol reduces the gastric damage induced by ethanol and indomethacin, at least in part, by mechanisms that involve endogenous prostaglandins, nitric oxide release, and or the activation of K(+)(ATP) channels.

  20. PKA-independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP.

    Science.gov (United States)

    Komai, Ali M; Brännmark, Cecilia; Musovic, Saliha; Olofsson, Charlotta S

    2014-12-01

    We examined the effects of cAMP, Ca(2+) and ATP on exocytosis and adipokine release in white adipocytes by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of adipokine secretion. 3T3-L1 adipocyte exocytosis proceeded even in the complete absence of intracellular Ca(2+) ([Ca(2+)]i; buffered with BAPTA) provided cAMP (0.1 mm) was included in the intracellular (pipette-filling) solution. Exocytosis typically plateaued within ∼10 min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3 mm ATP in combination with elevation of [Ca(2+)]i to ≥700 nm augmented the rate of cAMP-evoked exocytosis ∼2-fold and exocytosis proceeded for longer periods (≥20 min) than with cAMP alone. Exocytosis was stimulated to a similar extent upon substitution of cAMP by the Epac (exchange proteins activated by cAMP) agonist 8-Br-2'-O-Me-cAMP (1 mm included in the pipette solution). Inhibition of protein kinase A (PKA) by addition of Rp-cAMPS (0.5 mm) to the cAMP-containing pipette solution was without effect. A combination of the adenylate cyclase activator forskolin (10 μm) and the phosphodiesterase inhibitor IBMX (200 μm; forsk-IBMX) augmented adiponectin secretion measured over 30 min 3-fold and 2-fold in 3T3-L1 and human subcutaneous adipocytes, respectively. This effect was unaltered by pre-loading of cells with the Ca(2+) chelator BAPTA-AM and 2-fold amplified upon inclusion of the Ca(2+) ionophore ionomycin (1 μm) in the extracellular solution. Adiponectin release was also stimulated by the membrane-permeable Epac agonist 8-Br-2'-O-Me-cAMP-AM but unaffected by inclusion of the membrane-permeable PKA inhibitor Rp-8-Br-cAMPS (200 μm). The adipokines leptin, resistin and apelin were present in low amounts in the incubation medium (1-6% of measured adiponectin). Adipsin was secreted in substantial quantities (50% of adiponectin concentration) but release of this adipokine was unaffected by forsk

  1. NASA ATP Force Measurement Technology Capability Strategic Plan

    Science.gov (United States)

    Rhew, Ray D.

    2008-01-01

    The Aeronautics Test Program (ATP) within the National Aeronautics and Space Administration (NASA) Aeronautics Research Mission Directorate (ARMD) initiated a strategic planning effort to re-vitalize the force measurement capability within NASA. The team responsible for developing the plan included members from three NASA Centers (Langley, Ames and Glenn) as well as members from the Air Force s Arnold Engineering and Development Center (AEDC). After visiting and discussing force measurement needs and current capabilities at each participating facility as well as selected force measurement companies, a strategic plan was developed to guide future NASA investments. This paper will provide the details of the strategic plan and include asset management, organization and technology research and development investment priorities as well as efforts to date.

  2. Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in S. cerevisiae.

    Science.gov (United States)

    Karlin, Justin; Fischhaber, Paula L

    2013-06-01

    Several modes of eukaryotic of DNA double strand break repair (DSBR) depend on synapsis of complementary DNA. The Rad51 ATPase, the S. cerevisiae homolog of E. coli RecA, plays a key role in this process by catalyzing homology searching and strand exchange between an invading DNA strand and a repair template (e.g. sister chromatid or homologous chromosome). Synthesis dependent strand annealing (SDSA), a mode of DSBR, requires Rad51. Another repair enzyme, the Rad1-Rad10 endonuclease, acts in the final stages of SDSA, hydrolyzing 3' overhanging single-stranded DNA. Here we show in vivo by fluorescence microscopy that the ATP binding function of yeast Rad51 is required to recruit Rad10 SDSA sites indicating that Rad51 pre-synaptic filament formation must occur prior to the recruitment of Rad1-Rad10. Our data also show that Rad51 ATPase activity, an important step in Rad51 filament disassembly, is not absolutely required in order to recruit Rad1-Rad10 to DSB sites.

  3. Heterogeneity of ATP-sensitive K+ Channels in Cardiac Myocytes

    Science.gov (United States)

    Hong, Miyoun; Bao, Li; Kefaloyianni, Eirini; Agullo-Pascual, Esperanza; Chkourko, Halina; Foster, Monique; Taskin, Eylem; Zhandre,