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

Science.gov (United States)

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

2018-03-20

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

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

Science.gov (United States)

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

2013-04-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the γ-subunit present in tomato leaf and green fruit chloroplasts by the atypical γ-subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

Science.gov (United States)

He, Jiuya; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2017-08-22

The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

Cloning and characterization of ATP synthase CF1 α gene from ...

African Journals Online (AJOL)

ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-08-27

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

Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

Directory of Open Access Journals (Sweden)

Irene Mavelli

2012-02-01

Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg2+

International Nuclear Information System (INIS)

Du, Z.; Boyer, P.D.

1989-01-01

The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg 2+ with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg 2+ is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg 2+ released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg 2+ the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg 2+ and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg 2+ . The replacement of [ 3 H]ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg 2+ and ADP bound at one catalytic site without P i block catalysis by all three enzyme sites. The inhibited form induced by Mg 2+ and ADP may occur only under laboratory conditions and not have an in vivo role

Low energy costs of F1Fo ATP synthase reversal in colon carcinoma cells deficient in mitochondrial complex IV.

Science.gov (United States)

Zhdanov, Alexander V; Andreev, Dmitry E; Baranov, Pavel V; Papkovsky, Dmitri B

2017-05-01

Mitochondrial polarisation is paramount for a variety of cellular functions. Under ischemia, mitochondrial membrane potential (ΔΨm) and proton gradient (ΔpH) are maintained via a reversal of mitochondrial F1Fo ATP synthase (mATPase), which can rapidly deplete ATP and drive cells into energy crisis. We found that under normal conditions in cells with disassembled cytochrome c oxidase complex (COX-deficient HCT116), mATPase maintains ΔΨm at levels only 15-20% lower than in WT cells, and for this utilises relatively little ATP. For a small energy expenditure, mATPase enables mitochondrial ΔpH, protein import, Ca 2+ turnover, and supports free radical detoxication machinery enlarged to protect the cells from oxidative damage. Whereas in COX-deficient cells the main source of ATP is glycolysis, the ΔΨm is still maintained upon inhibition of the adenine nucleotide translocators with bongkrekic acid and carboxyatractyloside, indicating that the role of ANTs is redundant, and matrix substrate level phosphorylation alone or in cooperation with ATP-Mg/P i carriers can continuously support the mATPase activity. Intriguingly, we found that mitochondrial complex III is active, and it contributes not only to free radical production, but also to ΔΨm maintenance and energy budget of COX-deficient cells. Overall, this study demonstrates that F1Fo ATP synthase can support general mitochondrial and cellular functions, working in extremely efficient 'energy saving' reverse mode and flexibly recruiting free radical detoxication and ATP producing / transporting pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

African Journals Online (AJOL)

Yomi

2012-01-24

Jan 24, 2012 ... protein involved in temperature challenge in S. constricta. Key words: Sinonovacula constricta, ATP synthase, ... MATERIALS AND METHODS. Experimental animals. Sinonovacula constricta (7 to 8 g ... Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to ...

Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

KAUST Repository

Salunke, Rahul

2018-05-14

The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomer (~600 kDa) and dimer (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex will facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

KAUST Repository

Salunke, Rahul; Mourier, Tobias; Banerjee, Manidipa; Pain, Arnab; Shanmugam, Dhanasekaran

2018-01-01

The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomer (~600 kDa) and dimer (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex will facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

Science.gov (United States)

Seyfert, K.; Oosaka, T.; Yaginuma, H.; Ernst, S.; Noji, H.; Iino, R.; Börsch, M.

2011-03-01

FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

Science.gov (United States)

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

2017-03-28

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1 , ATP5G2 , and ATP5G3 , encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1 , ATP5G2 , and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

Class II recombinant phosphoribosyl diphosphate synthase from spinach

DEFF Research Database (Denmark)

Krath, B N; Hove-Jensen, B

2001-01-01

to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...... an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x...... mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6....

Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg sup 2+

Energy Technology Data Exchange (ETDEWEB)

Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

1989-01-24

The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg{sup 2+} with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg{sup 2+} is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg{sup 2+} released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg{sup 2+} the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg{sup 2+} and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg{sup 2+}. The replacement of ({sup 3}H)ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg{sup 2+} and ADP bound at one catalytic site without P{sub i} block catalysis by all three enzyme sites. The inhibited form induced by Mg{sup 2+} and ADP may occur only under laboratory conditions and not have an in vivo role.

Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

KAUST Repository

Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

2010-01-01

Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine.

Directory of Open Access Journals (Sweden)

Janny A Villa-Pulgarín

2017-08-01

Full Text Available Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated.Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast.The present study shows that the

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

KAUST Repository

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

2011-01-01

In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi→ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

Science.gov (United States)

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

2017-01-22

F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

Roberts, J.K.M.; Aubert, S.; Gout, E.; Bligny, R.; Douce, R.

1997-01-01

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

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

Science.gov (United States)

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

2016-12-01

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

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

Science.gov (United States)

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

2014-01-01

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

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

Science.gov (United States)

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

2016-04-01

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

ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

Directory of Open Access Journals (Sweden)

Kexiu Song

2014-01-01

Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

ATP Synthase Deficiency due to TMEM70 Mutation Leads to Ultrastructural Mitochondrial Degeneration and Is Amenable to Treatment

Directory of Open Access Journals (Sweden)

Anne K. Braczynski

2015-01-01

Full Text Available TMEM70 is involved in the biogenesis of mitochondrial ATP synthase and mutations in the TMEM70 gene impair oxidative phosphorylation. Herein, we report on pathology and treatment of ATP synthase deficiency in four siblings. A consanguineous family of Roma (Gipsy ethnic origin gave birth to 6 children of which 4 were affected presenting with dysmorphic features, failure to thrive, cardiomyopathy, metabolic crises, and 3-methylglutaconic aciduria as clinical symptoms. Genetic testing revealed a homozygous mutation (c.317-2A>G in the TMEM70 gene. While light microscopy was unremarkable, ultrastructural investigation of muscle tissue revealed accumulation of swollen degenerated mitochondria with lipid crystalloid inclusions, cristae aggregation, and exocytosis of mitochondrial material. Biochemical analysis of mitochondrial complexes showed an almost complete ATP synthase deficiency. Despite harbouring the same mutation, the clinical outcome in the four siblings was different. Two children died within 60 h after birth; the other two had recurrent life-threatening metabolic crises but were successfully managed with supplementation of anaplerotic amino acids, lipids, and symptomatic treatment during metabolic crisis. In summary, TMEM70 mutations can cause distinct ultrastructural mitochondrial degeneration and almost complete deficiency of ATP synthase but are still amenable to treatment.

Applications of new biophysical techniques to supramolecular structure of ATP synthase

International Nuclear Information System (INIS)

Zhu Jie; Wang Guodong

2007-01-01

The developing modern physical techniques offer a series of abundant and effective methods to study ATP synthase in structure and function. Firstly we stressed on the dialectic relationship between physical techniques and the improvement of science in history, and introduced a lot of physical techniques in common use in protein researches such as mass spectroscopy, nuclear magnetic resonance, synchronization X-ray diffraction, infrared spectroscopy and ultraviolet spectroscopy, and then reviewed their application status in quo to ATP synthase. Secondly we paid out attention to the burgeoning unconventionally instruments, i.e., the atomic force microscope and the fluorescence resonance energy transform (FRET) which have attracted the professional attention, and introduced latest application and researches' achievements. Compared the development of the techniques in recent years, we have set forth the shortcoming and excellence of all kinds of equipments introduced. And it was ended with the conclusion that it is necessary to manage the possible instruments effectively and sufficient for the personalities, and given out the optimum research routes which emphasized on the new techniques and novel methods, i.e., the atomic force microscope and FRET. (authors)

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

Directory of Open Access Journals (Sweden)

Sarah Schulz

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Assembly factors of F1FO-ATP synthase across genomes

Czech Academy of Sciences Publication Activity Database

Pícková, Andrea; Potocký, Martin; Houštěk, Josef

2005-01-01

Roč. 59, č. 3 (2005), s. 393-402 ISSN 0887-3585 R&D Projects: GA MŠk(CZ) 1M0520; GA MZd(CZ) NR7790 Grant - others:GA UK(CZ) 12/2002; GA UK(CZ) 11/2004; EC Framework Programme(XE) LSHM-CT-2004-503116 Institutional research plan: CEZ:AV0Z50110509 Keywords : assembly * ATP synthase * phylogenetic and sequence analysis Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.684, year: 2005

F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.

OpenAIRE

Lutter, R; Saraste, M; van Walraven, H S; Runswick, M J; Finel, M; Deatherage, J F; Walker, J E

1993-01-01

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20...

Bioenergetic Consequences of FLAG Tag Addition to the C-Terminus of Subunit 8 of Yeast Saccharomyces cerevisiae Mitochondrial ATP Synthase

Directory of Open Access Journals (Sweden)

I MADE ARTIKA

2010-09-01

Full Text Available The yeast mitochondrial F1F0-ATP synthase is a multisubunit complex that contains at least 17 different subunits. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial ATP8 gene. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. FLAG tag addition to subunit 8 protein potentially facilitate elucidation of its topology, structure, and function. It has been shown that following incorporation of FLAG tag to its C-terminus, subunit 8 still assemble into functional ATP synthase complex. In order to analyze bioenergetic consequences of the FLAG tag addition, a yeast strain expressing FLAG tagged-subunit 8 was subjected to cellular respiration assays. Results obtained showed that addition of FLAG tag to the C-terminus of subunit 8 does not impair its proper functioning. The FLAG tag system, therefore, can be employed to study subunit 8′s detailed structure, topology, and function.

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

Directory of Open Access Journals (Sweden)

Priscila Camillo Teixeira

2011-06-01

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

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

DEFF Research Database (Denmark)

Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose

2003-01-01

quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting...... synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins...

Energy Equivalence of Information in the Mitochondrion and the Thermodynamic Efficiency of ATP Synthase.

Science.gov (United States)

Matta, Chérif F; Massa, Lou

2015-09-01

Half a century ago, Johnson and Knudsen resolved the puzzle of the apparent low efficiency of the kidney (∼ 0.5%) compared to most other bodily organs (∼ 40%) by taking into account the entropic cost of ion sorting, the principal function of this organ. Similarly, it is shown that the efficiency of energy transduction of the chemiosmotic proton-motive force by ATP synthase is closer to 90% instead of the oft-quoted textbook value of only 60% when information theoretic considerations are applied to the mitochondrion. This high efficiency is consistent with the mechanical energy transduction of ATP synthase known to be close to the 100% thermodynamic limit. It would have been wasteful for evolution to maximize the mechanical energy transduction to 100% while wasting 40% of the chemiosmotic free energy in the conversion of the proton-motive force into mechanical work before being captured as chemical energy in adenosine 5'-triphosphate.

Functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid

International Nuclear Information System (INIS)

Rothenberg, M.; Hanson, M.R.

1988-01-01

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences using electrophoresis and autoradiography, indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts

Evolution of the F0F1 ATP synthase complex in light of the patchy distribution of different bioenergetic pathways across prokaryotes.

Directory of Open Access Journals (Sweden)

Vassiliki Lila Koumandou

2014-09-01

Full Text Available Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships. Wanting to address how this metabolic diversity evolved, we mapped the distribution of nine bioenergetic modes on a phylogenetic tree based on 16S rRNA sequences from 272 species representing the full diversity of prokaryotic lineages. This highlights the patchy distribution of many pathways across different lineages, and suggests either up to 26 independent origins or 17 horizontal gene transfer events. Next, we used comparative genomics and phylogenetic analysis of all subunits of the F0F1 ATP synthase, common to most bacterial lineages regardless of their bioenergetic mode. Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of atpI and of the beta subunit of the F0 subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits. We

The FoF1-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Zíková, Alena; Schnaufer, A.; Dalley, A. R.; Panigrahi, A. K.; Stuart, K. D.

2009-01-01

Roč. 5, č. 5 (2009), s. 1-15 E-ISSN 1932-6203 Keywords : mitochondrion * respiratory chain * / ATP synthase * TAP-tag * RNAi Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.351, year: 2009

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

Science.gov (United States)

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

2013-07-01

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

Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

KAUST Repository

Beke-Somfai, Tamás

2010-01-26

Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

International Nuclear Information System (INIS)

Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

2010-01-01

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

Oligomycin frames a common drug-binding site in the ATP synthase

Energy Technology Data Exchange (ETDEWEB)

Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

2015-12-01

We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

Science.gov (United States)

Ernst, Stefan; Düser, Monika G.; Zarrabi, Nawid; Börsch, Michael

2012-03-01

The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the γ and ɛ subunits of the F1 part. Because γ and ɛ subunits rotate in 120° steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Förster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations.

Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ∊

International Nuclear Information System (INIS)

Roy, Ankoor; Hutcheon, Marcus L.; Duncan, Thomas M.; Cingolani, Gino

2012-01-01

A phosphomimetic mutation in subunit ∊ dramatically increases reproducibility for crystallization of Escherichia coli ATP synthase catalytic complex (F 1 ) (subunit composition α 3 β 3 γ∊). Diffraction data were collected to ∼3.15 Å resolution using synchrotron radiation. The bacterial ATP synthase (F O F 1 ) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F O F 1 represents nature’s smallest rotary motor, composed of a membrane-embedded proton transporter (F O ) and a peripheral catalytic complex (F 1 ). The ATPase activity of isolated F 1 is fully expressed by the α 3 β 3 γ ‘core’, whereas single δ and ∊ subunits are required for structural and functional coupling of E. coli F 1 to F O . In contrast to mitochondrial F 1 -ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F 1 -ATPase catalytic core. Destabilizing the compact conformation of ∊’s C-terminal domain with a phosphomimetic mutation (∊S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F 1 that diffract to ∼3.15 Å resolution

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

KAUST Repository

Beke-Somfai, T.

2011-03-07

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

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

Czech Academy of Sciences Publication Activity Database

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

2004-01-01

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

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

Science.gov (United States)

de Kloet, S R

2001-08-01

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

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

KAUST Repository

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

2013-01-01

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

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

KAUST Repository

Beke-Somfai, T.

2013-01-23

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

Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen synthase by a multifunctional protein kinase (ATP-citrate lyase kinase)

International Nuclear Information System (INIS)

Sheorain, V.S.; Ramakrishna, S.; Benjamin, W.B.; Soderling, T.R.

1985-01-01

A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and]2number 2 PO 4 /mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the 34 PO 4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro

The assembly of F1FO-ATP synthase is disrupted upon interference of RNA editing in Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Hashimi, Hassan; Benkovičová, V.; Čermáková, P.; Lai, De Hua; Horváth, A.; Lukeš, Julius

2010-01-01

Roč. 40, č. 1 (2010), s. 45-54 ISSN 0020-7519 R&D Projects: GA ČR GA204/06/1558; GA AV ČR IAA500960705 Institutional research plan: CEZ:AV0Z60220518 Keywords : RNA editing * ATP synthase * mitochondrion * Trypanosoma * respiratory complex * membrane potential Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.822, year: 2010

Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

International Nuclear Information System (INIS)

Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

1991-01-01

Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

Directory of Open Access Journals (Sweden)

Luciana Galetto

Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

Science.gov (United States)

Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

2016-01-01

Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondria

International Nuclear Information System (INIS)

Silvester, Jocelyn A.; Kane Dickson, Veronica; Runswick, Michael J.; Leslie, Andrew G. W.; Walker, John E.

2006-01-01

A recombinant subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been crystallized and a native data set has been collected to 2.8 Å resolution. A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79–184 of subunit b, residues 1–124 of subunit d and the entire F 6 subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 Å resolution. They belong to the monoclinic space group P2 1

Simultaneous monitoring of the two coupled motors of a single FoF1-ATP synthase by three-color FRET using duty cycle-optimized triple-ALEX

Science.gov (United States)

Zarrabi, N.; Ernst, S.; Düser, M. G.; Golovina-Leiker, A.; Becker, W.; Erdmann, R.; Dunn, S. D.; Börsch, M.

2009-02-01

FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ɛ subunits of the F1 sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.

A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

Directory of Open Access Journals (Sweden)

Nevzat Selim Gokay

2016-01-01

Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice

Czech Academy of Sciences Publication Activity Database

Vrbacký, Marek; Kovalčíková, Jana; Chawengsaksophak, Kallayanee; Beck, Inken; Mráček, Tomáš; Nůsková, Hana; Sedmera, David; Papoušek, František; Kolář, František; Sobol, Margaryta; Hozák, Pavel; Sedláček, Radislav; Houštěk, Josef

2016-01-01

Roč. 25, č. 21 (2016), s. 4674-4685 ISSN 0964-6906 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MŠk(CZ) ED1.1.00/02.0109; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) LM2015062; GA MZd(CZ) NV16-33018A; GA MŠk(CZ) LM2015040 Institutional support: RVO:67985823 ; RVO:68378050 Keywords : mouse knockout * mitochondria * ATP synthase * TMEM70 * biogenesis * mitochondrial diseases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.340, year: 2016

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

Science.gov (United States)

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-06-18

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

International Nuclear Information System (INIS)

Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

2013-01-01

Highlights: ► ATP-treated sciatic explants shows the decreased expression of p75NGFR. ► Extracellular ATP inhibits the expression of phospho-ERK1/2. ► Lysosomal exocytosis is involved in Schwann cell dedifferentiation. ► Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition

Science.gov (United States)

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

2016-01-01

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

Quantum-mechanical analysis of amino acid residues function in the proton transport during F0F1-ATP synthase catalytic cycle

Science.gov (United States)

Ivontsin, L. A.; Mashkovtseva, E. V.; Nartsissov, Ya R.

2017-11-01

Implications of quantum-mechanical approach to the description of proton transport in biological systems are a tempting subject for an overlapping of fundamental physics and biology. The model of proton transport through the integrated membrane enzyme FoF1-ATP synthase responsible for ATP synthesis was developed. The estimation of the mathematical expectation of the proton transfer time through the half-channel was performed. Observed set of proton pathways through the inlet half-channel showed the nanosecond timescale highly dependable of some amino acid residues. There were proposed two types of crucial amino acids: critically localized (His245) and being a part of energy conserving system (Asp119).

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

Science.gov (United States)

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

2017-03-28

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

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

Science.gov (United States)

Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

2016-02-01

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. Published by Elsevier Inc.

Nuclear genetic defects of mitochondrial ATP synthase

Czech Academy of Sciences Publication Activity Database

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

2014-01-01

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

Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro.

Science.gov (United States)

Caiazzo, Elisabetta; Tedesco, Idolo; Spagnuolo, Carmela; Russo, Gian Luigi; Ialenti, Armando; Cicala, Carla

2016-06-01

Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP--and ADPase activity in rat platelets.

Prostaglandin E(2) synthase inhibition as a therapeutic target.

Science.gov (United States)

Iyer, Jitesh P; Srivastava, Punit K; Dev, Rishabh; Dastidar, Sunanda G; Ray, Abhijit

2009-07-01

Most NSAIDs function by inhibiting biosynthesis of PGE(2) by inhibition of COX-1 and/or COX-2. Since COX-1 has a protective function in the gastro-intestinal tract (GIT), non-selective inhibition of both cycloxy genases leads to moderate to severe gastro-intestinal intolerance. Attempts to identify selective inhibitors of COX-2, led to the identification of celecoxib and rofecoxib. However, long-term use of these drugs has serious adverse effects of sudden myocardial infarction and thrombosis. Drug-mediated imbalance in the levels of prostaglandin I(2) (PGI(2)) and thromboxane A(2) (TXA(2)) with a bias towards TXA(2) may be the primary reason for these events. This resulted in the drugs being withdrawn from the market, leaving a need for an effective and safe anti-inflammatory drug. Recently, the focus of research has shifted to enzymes downstream of COX in the prosta glandin biosynthetic pathway such as prostaglandin E(2) synthases. Microsomal prostaglandin E(2) synthase-1 (mPGES-1) specifically isomerizes PGH(2) to PGE(2), under inflammatory conditions. In this review, we examine the biology of mPGES-1 and its role in disease. Progress in designing molecules that can selectively inhibit mPGES-1 is reviewed. mPGES-1 has the potential to be a target for anti-inflammatory therapy, devoid of adverse GIT and cardiac effects and warrants further investigation.

Trimeric form of intracellular ATP synthase subunit β of Aggregatibacter actinomycetemcomitans binds human interleukin-1β.

Directory of Open Access Journals (Sweden)

Annamari Paino

Full Text Available Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.

The process of lipid storage in insect oocytes: The involvement of β-chain of ATP synthase in lipophorin-mediated lipid transfer in the chagas' disease vector Panstrongylus megistus (Hemiptera: Reduviidae).

Science.gov (United States)

Fruttero, Leonardo L; Leyria, Jimena; Ramos, Fabián O; Stariolo, Raúl; Settembrini, Beatriz P; Canavoso, Lilián E

2017-01-01

Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic β chain of ATP synthase (β-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of β-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of β-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of β-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and β-ATPase. In vivo experiments with an anti-β-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that β-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus

Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor

OpenAIRE

Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

2015-01-01

Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits P. falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report 3 crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all 3 structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of...

Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

Science.gov (United States)

Zhang, Conggang; Liu, Zeyu; Bunker, Eric; Ramirez, Adrian; Lee, Schuyler; Peng, Yinghua; Tan, Aik-Choon; Eckhardt, S Gail; Chapnick, Douglas A; Liu, Xuedong

2017-09-08

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP Synthase

Science.gov (United States)

Stan-Lotter, Helga; Hochstein, Lawrence I.

1989-01-01

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F sub 1 moiety from the Escherichia coli ATP Synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa, and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20 to 22 Mol percent). Peptide mapping of sodium dodecylsulfate-denatured subunits I and II showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F sub 1 ATPase (EC 3.6.1.34) from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, Halobacteria in general, possess an ATPase which is unlike the ubiquitous F sub o F sub 1 - ATP Synthase.

Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

Energy Technology Data Exchange (ETDEWEB)

Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha; Xu, Yao; Qin, Ximing; Johnson, Carl H. (Vanderbilt)

2014-10-02

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, such that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P{sub i} (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the

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

Directory of Open Access Journals (Sweden)

Hayoz Sébastien

2012-05-01

Full Text Available Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP

Compensatory upregulation of respiratory chain complexes III and IV in isolated deficiency of ATP synthase due to TMEM70 mutation

Czech Academy of Sciences Publication Activity Database

Karbanová-Havlíčková, Vendula; Vrbacká-Čížková, Alena; Hejzlarová, Kateřina; Nůsková, Hana; Stránecký, V.; Potocká, Andrea; Kmoch, S.; Houštěk, Josef

2012-01-01

Roč. 1817, č. 7 (2012), s. 1037-1043 ISSN 0005-2728 R&D Projects: GA MZd(CZ) NS9759; GA ČR(CZ) GAP303/11/0970; GA ČR(CZ) GD303/03/H065; GA MŠk(CZ) 1M0520 Grant - others:Univerzita Karlova(CZ) 370411 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATP synthase * TMEM70 * disease * gene expression profiling * oxidative phosphorylation * mitochondrial biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.624, year: 2012

Genetické a funkční příčiny mitochondriálních chorob vyvolaných defekty ATP syntázy

OpenAIRE

Tauchmannová, Kateřina

2015-01-01

Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency....

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

Science.gov (United States)

1986-01-01

Paddle and Burnstock (326), Williams and Forrester (463), Forrester and Williams (151) and Clemens and Forrester (82) provide evidence that hypoxia may...an ATp4 - receptor. Fed. Proc. 45:208, 1986. (abstr) 99. Dahlen , S.E. and Hedqvist, P. ATP, B,y-methylene ATP andN adenosine inhibit non-cholinergic...regulation of skeletal muscle blood low. Circ Res. 29:375-384, 1971. 117. Dodd, J., Jahr, C.E., Hamilton, P.N., Heath, M.J., Matthew , W.P., and Jessell, T.M

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

Science.gov (United States)

Liu, Sidong; Charlesworth, Thomas J; Bason, John V; Montgomery, Martin G; Harbour, Michael E; Fearnley, Ian M; Walker, John E

2015-05-15

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

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

Science.gov (United States)

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

2003-09-01

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

Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

Directory of Open Access Journals (Sweden)

Brian Panicucci

2017-04-01

Full Text Available The mitochondrial (mt FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF, but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF, which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm. Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1 binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1, but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

DEFF Research Database (Denmark)

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

2016-01-01

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects...... of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 μM)>celecoxib (IC50: 14.92 ± 6.40 μM)>valdecoxib (IC50: 161.4 ± 28.6 μM)>rofecoxib (IC50...... correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs...

Alteration of structure and function of ATP synthase and cytochrome c oxidase by lack of F-o-a and Cox3 subunits caused by mitochondrial DNA 9205delTA mutation

Czech Academy of Sciences Publication Activity Database

Hejzlarová, Kateřina; Kaplanová, Vilma; Nůsková, Hana; Kovářová, Nikola; Ješina, Pavel; Drahota, Zdeněk; Mráček, Tomáš; Seneca, S.; Houštěk, Josef

2015-01-01

Roč. 466, č. 3 (2015), s. 601-611 ISSN 0264-6021 R&D Projects: GA ČR(CZ) GAP303/11/0970; GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA ČR(CZ) GAP303/12/1363 Institutional support: RVO:67985823 Keywords : ATP synthase * cytochrome c oxidase * mitochondrial diseases * mtDNA MT-ATP6 mutation * oxidative phosphorylation * threshold effect Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.562, year: 2015

Mechanism of Action and Inhibition of dehydrosqualene Synthase

Energy Technology Data Exchange (ETDEWEB)

F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

2011-12-31

'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

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

DEFF Research Database (Denmark)

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

2013-01-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active...... synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes...... in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report...

Imidazopyridine Compounds Inhibit Mycobacterial Growth by Depleting ATP Levels.

Science.gov (United States)

O'Malley, Theresa; Alling, Torey; Early, Julie V; Wescott, Heather A; Kumar, Anuradha; Moraski, Garrett C; Miller, Marvin J; Masquelin, Thierry; Hipskind, Philip A; Parish, Tanya

2018-06-01

The imidazopyridines are a promising new class of antitubercular agents with potent activity in vitro and in vivo We isolated mutants of Mycobacterium tuberculosis resistant to a representative imidazopyridine; the mutants had large shifts (>20-fold) in MIC. Whole-genome sequencing revealed mutations in Rv1339, a hypothetical protein of unknown function. We isolated mutants resistant to three further compounds from the series; resistant mutants isolated from two of the compounds had single nucleotide polymorphisms in Rv1339 and resistant mutants isolated from the third compound had single nucleotide polymorphisms in QcrB, the proposed target for the series. All the strains were resistant to two compounds, regardless of the mutation, and a strain carrying the QcrB T313I mutation was resistant to all of the imidazopyridine derivatives tested, confirming cross-resistance. By monitoring pH homeostasis and ATP generation, we confirmed that compounds from the series were targeting QcrB; imidazopyridines disrupted pH homeostasis and depleted ATP, providing further evidence of an effect on the electron transport chain. A representative compound was bacteriostatic against replicating bacteria, consistent with a mode of action against QcrB. The series had a narrow inhibitory spectrum, with no activity against other bacterial species. No synergy or antagonism was seen with other antituberculosis drugs under development. In conclusion, our data support the hypothesis that the imidazopyridine series functions by reducing ATP generation via inhibition of QcrB. Copyright © 2018 O'Malley et al.

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

DEFF Research Database (Denmark)

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

2016-01-01

in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen...... and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria...

Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells.

Science.gov (United States)

Manning Fox, Jocelyn E; Karaman, Gunce; Wheeler, Michael B

2006-11-17

Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.

Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator

Directory of Open Access Journals (Sweden)

Satoshi Miyamoto

2016-05-01

Full Text Available AMP-activated protein kinase (AMPK is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0 accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0 significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0 could be responsible for the enhanced ATP production via activation of the glycolytic pathway.

Mitochondrial dysfunction is responsible for fatty acid synthase inhibition-induced apoptosis in breast cancer cells by PdpaMn.

Science.gov (United States)

Wang, Qiang; Du, Xia; Zhou, Bingjie; Li, Jing; Lu, Wenlong; Chen, Qiuyun; Gao, Jing

2017-12-01

Targeting cellular metabolism is becoming a hallmark to overcome drug resistance in breast cancer treatment. Activation of fatty acid synthase (FASN) has been shown to promote breast cancer cell growth. However, there is no concrete report underlying the mechanism associated with mitochondrial dysfunction in relation to fatty acid synthase inhibition-induced apoptosis in breast cancer cells. The current study is aimed at exploring the effect of the novel manganese (Mn) complex, labeled as PdpaMn, on lipid metabolism and mitochondrial function in breast cancer cells. Herein, we observed that PdpaMn displayed strong cytotoxicity on breast cancer cell lines and selectively targeted the tumor without affecting the normal organs or cells in vivo. We also observed that PdpaMn could bind to TE domain of FASN and decrease the activity and the level of expression of FASN, which is an indication that FASN could serve as a target of PdpaMn. In addition, we demonstrated that PdpaMn increased intrinsic apoptosis in breast cancer cells relayed by a suppressed the level of expression of FASN, followed by the release of mitochondrial cytochrome c and the activation of caspases-9. Instigated by the above observations, we hypothesized that PdpaMn-induced apoptosis events are dependent on mitochondrial dysfunction. Indeed, we found that mitochondrial membrane potential (MMP) collapse, mitochondrial oxygen consumption reduction and adenosine triphosphate (ATP) release were deeply repressed. Furthermore, our results showed that PdpaMn significantly increased the reactive oxygen species (ROS) production, and the protection conferred by the free radical scavenger N-acetyl-cysteine (NAC) indicates that PdpaMn-induced apoptosis through an oxidative stress-associated mechanism. More so, the above results have demonstrated that mitochondrial dysfunction participated in FASN inhibition-induce apoptosis in breast cancer cells by PdpaMn. Therefore, PdpaMn may be considered as a good candidate

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

International Nuclear Information System (INIS)

Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

2011-01-01

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

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

Science.gov (United States)

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

2014-01-01

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

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

Directory of Open Access Journals (Sweden)

Ulyana Lalo

2014-01-01

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

Evidence for Fumonisin inhibition of ceramide synthase in humans: validation in follow-up studies in Guatemala

Science.gov (United States)

Fumonisins (FB) are mycotoxins found in corn. FB1 is the most common FB. It is the cause of farm animal diseases and is carcinogenic in rodents. The mode of action is the inhibition of ceramide synthase (CerS). Inhibition of CerS in mice causes a dose-dependent accumulation of sphinganine 1-phosphat...

( Atp9) gene between cytoplasmic male sterile line and its ...

African Journals Online (AJOL)

Administrator

2011-09-07

Sep 7, 2011 ... Soybean Research Institute of Nanjing Agricultural University, National Center for Soybean .... All these transgenic experiments confirmed the correlation ... editing of ATP synthase subunit 9 mRNA using wheat mitochondrial.

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells.

Science.gov (United States)

Santo-Domingo, Jaime; Chareyron, Isabelle; Broenimann, Charlotte; Lassueur, Steve; Wiederkehr, Andreas

2017-08-15

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed

Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Science.gov (United States)

Hammann, Eva; Zappe, Andrea; Keis, Stefanie; Ernst, Stefan; Matthies, Doreen; Meier, Thomas; Cook, Gregory M.; Börsch, Michael

2012-02-01

Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.

Vascular ATP-sensitive potassium channels are over-expressed and partially regulated by nitric oxide in experimental septic shock.

Science.gov (United States)

Collin, Solène; Sennoun, Nacira; Dron, Anne-Gaëlle; de la Bourdonnaye, Mathilde; Montemont, Chantal; Asfar, Pierre; Lacolley, Patrick; Meziani, Ferhat; Levy, Bruno

2011-05-01

To study the activation and expression of vascular (aorta and small mesenteric arteries) potassium channels during septic shock with or without modulation of the NO pathway. Septic shock was induced in rats by peritonitis. Selective inhibitors of vascular K(ATP) (PNU-37883A) or BK(Ca) [iberiotoxin (IbTX)] channels were used to demonstrate their involvement in vascular hyporeactivity. Vascular response to phenylephrine was measured on aorta and small mesenteric arteries mounted on a wire myograph. Vascular expression of potassium channels was studied by PCR and Western blot, in the presence or absence of 1400W, an inducible NO synthase (iNOS) inhibitor. Aortic activation of the transcriptional factor nuclear factor-kappaB (NF-κB) was assessed by electrophoretic mobility shift assay. Arterial pressure as well as in vivo and ex vivo vascular reactivity were reduced by sepsis and improved by PNU-37883A but not by IbTX. Sepsis was associated with an up-regulation of mRNA and protein expression of vascular K(ATP) channels, while expression of vascular BK(Ca) channels remained unchanged. Selective iNOS inhibition blunted the sepsis-induced increase in aortic NO, decreased NF-κB activation, and down-regulated vascular K(ATP) channel expression. Vascular K(ATP) but not BK(Ca) channels are activated, over-expressed, and partially regulated by NO via NF-κB activation during septic shock. Their selective inhibition restores arterial pressure and vascular reactivity and decreases lactate concentration. The present data suggest that selective vascular K(ATP) channel inhibitors offer potential therapeutic perspectives for septic shock.

ATP-independent DNA synthesis in Vaccinia-infected L cells

International Nuclear Information System (INIS)

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

1978-01-01

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

L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

Science.gov (United States)

Nakatsu, Daiki; Horiuchi, Yuta; Kano, Fumi; Noguchi, Yoshiyuki; Sugawara, Taichi; Takamoto, Iseki; Kubota, Naoto; Kadowaki, Takashi; Murata, Masayuki

2015-03-10

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

Identification of a new Mpl-interacting protein, Atp5d.

Science.gov (United States)

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

2014-06-01

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

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

Energy Technology Data Exchange (ETDEWEB)

Manceur, Aziza P. [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Tseng, Michael [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Holowacz, Tamara [Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Witterick, Ian [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Department of Otolaryngology, Head and Neck Surgery, University of Toronto, ON (Canada); Weksberg, Rosanna [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); McCurdy, Richard D. [The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); Warsh, Jerry J. [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Audet, Julie, E-mail: julie.audet@utoronto.ca [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada)

2011-09-10

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

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

Directory of Open Access Journals (Sweden)

Rini Widayanti

2013-07-01

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

Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

Science.gov (United States)

Walpole, Thomas B; Palmer, David N; Jiang, Huibing; Ding, Shujing; Fearnley, Ian M; Walker, John E

2015-04-01

The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria. © 2015 by The American

Cardamonin, a schistosomicidal chalcone from Piper aduncum L. (Piperaceae) that inhibits Schistosoma mansoni ATP diphosphohydrolase.

Science.gov (United States)

de Castro, Clarissa C B; Costa, Poliana S; Laktin, Gisele T; de Carvalho, Paulo H D; Geraldo, Reinaldo B; de Moraes, Josué; Pinto, Pedro L S; Couri, Mara R C; Pinto, Priscila de F; Da Silva Filho, Ademar A

2015-09-15

Schistosomiasis is one of the world's major public health problems, and praziquantel (PZQ) is the only available drug to treat this neglected disease with an urgent demand for new drugs. Recent studies indicated that extracts from Piper aduncum L. (Piperaceae) are active against adult worms of Schistosoma mansoni, the major etiological agent of human schistosomiasis. We investigated the in vitro schistosomicidal activity of cardamonin, a chalcone isolated from the crude extract of P. aduncum. Also, this present work describes, for the first time, the S. mansoni ATP diphosphohydrolase inhibitory activity of cardamonin, as well as, its molecular docking with S. mansoni ATPDase1, in order to investigate its mode of inhibition. In vitro schistosomicidal assays and confocal laser scanning microscopy were used to evaluate the effects of cardamonin on adult schistosomes. Cell viability was measured by MTT assay, and the S. mansoni ATPase activity was determined spectrophotometrically. Identification of the cardamonin binding site and its interactions on S. mansoni ATPDase1 were made by molecular docking experiments. A bioguided fractionation of the crude extract of P. aduncum was carried out, leading to identification of cardamonin as the active compound, along with pinocembrin and uvangoletin. Cardamonin (25, 50, and 100 µM) caused 100% mortality, tegumental alterations, and reduction of oviposition and motor activity of all adult worms of S. mansoni, without affecting mammalian cells. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner. Cardamonin also inhibited S. mansoni ATP diphosphohydrolase (IC50 of 23.54 µM). Molecular docking studies revealed that cardamonin interacts with the Nucleotide-Binding of SmATPDase 1. The nature of SmATPDase 1-cardamonin interactions is mainly hydrophobic and hydrogen bonding. This report provides evidence for the in vitro

Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI).

Science.gov (United States)

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

2016-03-01

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half maximal rate of inactivation (KI) of 7.69 ± 2.48 μM with Kinact/KI ratio of 0.343 min(-1) μM(-1). Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

DEFF Research Database (Denmark)

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

2009-01-01

ATP and NE concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and norepinephrine (NE) concentrations were measured during: 1) femoral arterial ATP infusion (0......, respectively (Pcontracting muscle (Pmuscle, whereas interstitial NE concentrations increased similarly in both active...... and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial prinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its...

HAEM SYNTHASE AND COBALT PORPHYRIN SYNTHASE IN VARIOUS MICRO-ORGANISMS.

Science.gov (United States)

PORRA, R J; ROSS, B D

1965-03-01

1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co(2+) ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co(2+) ions into these two porphyrins is rapid. These extracts are unable to insert Mn(2+), Zn(2+), Mg(2+) or Cu(2+) ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co(2+) and Fe(2+) ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co(2+) ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.

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).

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

Directory of Open Access Journals (Sweden)

Ronglan Zhao

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

The NMR solution structure of Mycobacterium tuberculosis F-ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine.

Science.gov (United States)

Joon, Shin; Ragunathan, Priya; Sundararaman, Lavanya; Nartey, Wilson; Kundu, Subhashri; Manimekalai, Malathy S S; Bogdanović, Nebojša; Dick, Thomas; Grüber, Gerhard

2018-03-01

Mycobacterium tuberculosis (Mt) F 1 F 0 ATP synthase (α 3 :β 3 :γ:δ:ε:a:b:b':c 9 ) is essential for the viability of growing and nongrowing persister cells of the pathogen. Here, we present the first NMR solution structure of Mtε, revealing an N-terminal β-barrel domain (NTD) and a C-terminal domain (CTD) composed of a helix-loop-helix with helix 1 and -2 being shorter compared to their counterparts in other bacteria. The C-terminal amino acids are oriented toward the NTD, forming a domain-domain interface between the NTD and CTD. The Mtε structure provides a novel mechanistic model of coupling c-ring- and ε rotation via a patch of hydrophobic residues in the NTD and residues of the CTD to the bottom of the catalytic α 3 β 3 -headpiece. To test our model, genome site-directed mutagenesis was employed to introduce amino acid changes in these two parts of the epsilon subunit. Inverted vesicle assays show that these mutations caused an increase in ATP hydrolysis activity and a reduction in ATP synthesis. The structural and enzymatic data are discussed in light of the transition mechanism of a compact and extended state of Mtε, which provides the inhibitory effects of this coupling subunit inside the rotary engine. Finally, the employment of these data with molecular docking shed light into the second binding site of the drug Bedaquiline. Structural data are available in the PDB under the accession number 5YIO. © 2018 Federation of European Biochemical Societies.

Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

DEFF Research Database (Denmark)

Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank

2011-01-01

CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer...... completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5 Å resolution...

Inhibition of fatty acid synthase prevents preadipocyte differentiation

International Nuclear Information System (INIS)

Schmid, Bernhard; Rippmann, Joerg F.; Tadayyon, Moh; Hamilton, Bradford S.

2005-01-01

Inhibition of fatty acid synthase (FAS) reduces food intake in rodents. As adipose tissue expresses FAS, we sought to investigate the effect of reduced FAS activity on adipocyte differentiation. FAS activity was suppressed either pharmacologically or by siRNA during differentiation of 3T3-L1 cells. Cerulenin (10 μM), triclosan (50 μM), and C75 (50 μM) reduced dramatically visible lipid droplet accumulation, while incorporation of [1- 14 C]acetate into lipids was reduced by 75%, 70%, and 90%, respectively. Additionally, the substances reduced FAS, CEBPα, and PPARγ mRNA by up to 85% compared to that of control differentiated cells. Transient transfection with FAS siRNA suppressed FAS mRNA and FAS activity, and this was accompanied by reduction of CEBPα and PPARγ mRNA levels, and complete prevention of lipid accumulation. CD36, a late marker of differentiation, was also reduced. Together, these results suggest that FAS generated signals may be essential to support preadipocyte differentiation

Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses

Directory of Open Access Journals (Sweden)

Geneviève Pèépin

2017-10-01

Full Text Available Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1 by low-dose camptothecin (CPT can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40 large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development.

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

Science.gov (United States)

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

2017-08-01

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

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

Directory of Open Access Journals (Sweden)

Masaki Nakano

2017-08-01

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

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

Science.gov (United States)

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

2013-03-01

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

Binding of ATP by pertussis toxin and isolated toxin subunits

International Nuclear Information System (INIS)

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

1990-01-01

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

Binding of ATP by pertussis toxin and isolated toxin subunits

Energy Technology Data Exchange (ETDEWEB)

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

1990-07-03

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

Inhibition by sodium nitroprusside of the expression of inducible nitric oxide synthase in rat neutrophils.

OpenAIRE

Mariotto, S; Cuzzolin, L; Adami, A; Del Soldato, P; Suzuki, H; Benoni, G

1995-01-01

A well-known nitric oxide (NO)-releasing compound, sodium nitroprusside (SNP), decreases in a dose-dependent manner NO synthase (NOS) activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS). This inhibitory action of SNP seems not to be due to its direct effect on the enzyme activity. The strong nitrosonium ion (NO+) character of SNP could be responsible for its inhibition of NOS induction in neutrophils.

Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

Science.gov (United States)

Thupari, J N; Pinn, M L; Kuhajda, F P

2001-07-13

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy. Copyright 2001 Academic Press.

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

Science.gov (United States)

Abramochkin, Denis V; Vornanen, Matti

2014-09-01

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

Chronic inhibition of nitric oxide synthase augments the ACTH response to exercise.

Science.gov (United States)

Jankord, Ryan; McAllister, Richard M; Ganjam, Venkataseshu K; Laughlin, M Harold

2009-03-01

Exercise can activate the hypothalamo-pituitary-adrenocortical (HPA) axis, and regular exercise training can impact how the HPA axis responds to stress. The mechanism by which acute exercise induces HPA activity is unclear. Therefore, the purpose of this study was to test the hypothesis that nitric oxide modulates the neuroendocrine component of the HPA axis during exercise. Female Yucatan miniature swine were treated with N-nitro-l-arginine methyl ester (l-NAME) to test the effect of chronic nitric oxide synthase (NOS) inhibition on the ACTH response to exercise. In addition, we tested the effect of NOS inhibition on blood flow to tissues of the HPA axis and report the effects of handling and treadmill exercise on the plasma concentrations of ACTH and cortisol. Chronic NOS inhibition decreased plasma NO(x) levels by 44%, increased mean arterial blood pressure by 46%, and increased expression of neuronal NOS in carotid arteries. Vascular conductance was decreased in the frontal cortex, the hypothalamus, and the adrenal gland. Chronic NOS inhibition exaggerated the ACTH response to exercise. In contrast, chronic NOS inhibition decreased the ACTH response to restraint, suggesting that the role of NO in modulating HPA activity is stressor dependent. These results demonstrate that NOS activity modulates the response of the neuroendocrine component of the HPA axis during exercise stress.

Phosphorylation of glyoxysomal malate synthase from castor oil seed endosperm and cucumber cotyledon

International Nuclear Information System (INIS)

Yang, Y.P; Randall, D.D.

1989-01-01

Glyoxysomal malate synthase (MS) was purified to apparent homogeneity from 3-d germinating castor oil seed endosperm by a relatively simple procedure including two sucrose density gradient centrifugations. Antibodies raised to the caster oil seed MS crossreacted with MS from cucumber cotyledon. MS was phosphorylated in both tissues in an MgATP dependent reaction. The phosphorylation pattern was similar for both enzymes and both enzymes were inhibited by NaF, NaMo, (NH 4 )SO 4 , glyoxylate and high concentration of MgCl 2 (60 mM), but was not inhibited by NaCl and malate. Further characterization of the phosphorylation of MS from castor oil seed endosperms showed that the 5S form of MS is the form which is labelled by 32 P. The addition of exogenous alkaline phosphatase to MS not only decreased enzyme activity, but could also dephosphorylate phospho-MS. The relationship between dephosphorylation of MS and the decrease of MS activity is currently under investigation

Inhibition by sodium nitroprusside of the expression of inducible nitric oxide synthase in rat neutrophils.

Science.gov (United States)

Mariotto, S; Cuzzolin, L; Adami, A; Del Soldato, P; Suzuki, H; Benoni, G

1995-01-01

A well-known nitric oxide (NO)-releasing compound, sodium nitroprusside (SNP), decreases in a dose-dependent manner NO synthase (NOS) activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS). This inhibitory action of SNP seems not to be due to its direct effect on the enzyme activity. The strong nitrosonium ion (NO+) character of SNP could be responsible for its inhibition of NOS induction in neutrophils. PMID:7542530

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

Science.gov (United States)

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

2013-09-18

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

Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels.

NARCIS (Netherlands)

Graaf, A.O. de; Meijerink, J.P.P.; Heuvel, L.P.W.J. van den; Abreu, R.A. de; Witte, T.J.M. de; Jansen, J.H.; Smeitink, J.A.M.

2002-01-01

Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production

The consequences of long-term glycogen synthase kinase-3 inhibition on normal and insulin resistant rat hearts.

Science.gov (United States)

Flepisi, T B; Lochner, Amanda; Huisamen, Barbara

2013-10-01

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase, discovered as a regulator of glycogen synthase. GSK-3 may regulate the expression of SERCA-2a potentially affecting myocardial contractility. It is known to phosphorylate and inhibit IRS-1, thus disrupting insulin signalling. This study aimed to determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether chronic GSK-3 inhibition can prevent or reverse this. Weight matched male Wistar rats were rendered obese by hyperphagia using a special diet (DIO) for 16 weeks and compared to chow fed controls. Half of each group was treated with the GSK-3 inhibitor CHIR118637 (30 mg/kg/day) from week 12 to16 of the diet period. Biometric and biochemical parameters were measured and protein expression determined by Western blotting and specific antibodies. Ca(2+)ATPase activity was determined spectrophotometrically. Cardiomyocytes were prepared by collagenase perfusion and insulin stimulated 2-deoxy-glucose uptake determined. DIO rats were significantly heavier than controls, associated with increased intra-peritoneal fat and insulin resistance. GSK-3 inhibition did not affect weight but improved insulin resistance, also on cellular level. It had no effect on GSK-3 expression but elevated its phospho/total ratio and elevated IRS-2 expression. Obesity lowered SERCA-2a expression and activity while GSK-3 inhibition alleviated this. The phospho/total ratio of phospholamban underscored inhibition of SERCA-2a in obesity. In addition, signs of myocardial hypertrophy were observed in treated control rats. GSK-3 inhibition could not reverse all the detrimental effects of obesity but may be harmful in normal rat hearts. It regulates IRS-2, SERCA-2a and phospholamban expression but not IRS-1.

Inhibition of thromboxane synthase induces lung cancer cell death via increasing the nuclear p27

Energy Technology Data Exchange (ETDEWEB)

Leung, Kin Chung; Hsin, Michael K.Y.; Chan, Joey S.Y.; Yip, Johnson H.Y.; Li, Mingyue; Leung, Billy C.S. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Mok, Tony S.K. [Department of Clinical Oncology, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Warner, Timothy D. [The William Harvey Research Institute, Queen Mary University of London, London (United Kingdom); Underwood, Malcolm J. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Chen, George G., E-mail: gchen@cuhk.edu.hk [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong)

2009-10-15

The role of thromboxane in lung carcinogenesis is not clearly known, though thromboxane B2 (TXB{sub 2}) level is increased and antagonists of thromboxane receptors or TXA2 can induce apoptosis of lung cancer cells. p27, an atypical tumor suppressor, is normally sequestered in the nucleus. The increased nuclear p27 may result in apoptosis of tumor cells. We hypothesize that the inhibition of thromboxane synthase (TXS) induces the death of lung cancer cells and that such inhibition is associated with the nuclear p27 level. Our experiment showed that the inhibition of TXS significantly induced the death or apoptosis in lung cancer cells. The activity of TXS was increased in lung cancer. The nuclear p27 was remarkably reduced in lung cancer tissues. The inhibition of TXS caused the cell death and apoptosis of lung cancer cells, likely via the elevation of the nuclear p27 since the TXS inhibition promoted the nuclear p27 level and the inhibition of p27 by its siRNA recovered the cell death induced by TXS inhibition. Collectively, lung cancer cells produce high levels of TXB{sub 2} but their nuclear p27 is markedly reduced. The inhibition of TXS results in the p27-related induction of cell death in lung cancer cells.

Inhibition of thromboxane synthase induces lung cancer cell death via increasing the nuclear p27

International Nuclear Information System (INIS)

Leung, Kin Chung; Hsin, Michael K.Y.; Chan, Joey S.Y.; Yip, Johnson H.Y.; Li, Mingyue; Leung, Billy C.S.; Mok, Tony S.K.; Warner, Timothy D.; Underwood, Malcolm J.; Chen, George G.

2009-01-01

The role of thromboxane in lung carcinogenesis is not clearly known, though thromboxane B2 (TXB 2 ) level is increased and antagonists of thromboxane receptors or TXA2 can induce apoptosis of lung cancer cells. p27, an atypical tumor suppressor, is normally sequestered in the nucleus. The increased nuclear p27 may result in apoptosis of tumor cells. We hypothesize that the inhibition of thromboxane synthase (TXS) induces the death of lung cancer cells and that such inhibition is associated with the nuclear p27 level. Our experiment showed that the inhibition of TXS significantly induced the death or apoptosis in lung cancer cells. The activity of TXS was increased in lung cancer. The nuclear p27 was remarkably reduced in lung cancer tissues. The inhibition of TXS caused the cell death and apoptosis of lung cancer cells, likely via the elevation of the nuclear p27 since the TXS inhibition promoted the nuclear p27 level and the inhibition of p27 by its siRNA recovered the cell death induced by TXS inhibition. Collectively, lung cancer cells produce high levels of TXB 2 but their nuclear p27 is markedly reduced. The inhibition of TXS results in the p27-related induction of cell death in lung cancer cells.

Glycogen synthase activation by sugars in isolated hepatocytes.

Science.gov (United States)

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

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

Science.gov (United States)

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

2001-07-01

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

Electron transport phosphorylation in rumen butyrivibrios: unprecedented ATP yield for glucose fermentation to butyrate

Directory of Open Access Journals (Sweden)

Timothy eHackmann

2015-06-01

Full Text Available From a genomic analysis of rumen butyrivibrios (Butyrivibrio and Pseudobutyrivibrio spp., we have re-evaluated the contribution of electron transport phosphorylation to ATP formation in this group. This group is unique in that most (76% genomes were predicted to possess genes for both Ech and Rnf transmembrane ion pumps. These pumps act in concert with the NifJ and Bcd-Etf to form a electrochemical potential (ΔμH+ and ΔμNa+, which drives ATP synthesis by electron transport phosphorylation. Of the 62 total butyrivibrio genomes currently available from the Hungate 1000 project, all 62 were predicted to possess NifJ, which reduces oxidized ferredoxin (Fdox during pyruvate conversion to acetyl-CoA. All 62 possessed all subunits of Bcd-Etf, which reduces Fdox and oxidizes reduced NAD (NADred during crotonyl-CoA reduction. Additionally, 61 genomes possessed all subunits of the Rnf, which generates ΔμH+ or ΔμNa+ from oxidation of reduced Fd and reduction of oxidized NAD (NADox. Further, 47 genomes possessed all 6 subunits of the Ech, which generates ΔμH+ from oxidation of reduced Fd (Fdred. For glucose fermentation to butyrate and H2, the electrochemical potential established should drive synthesis of ~1.5 ATP by the F0F1-ATP synthase (possessed by all 62 genomes. The total yield is ~4.5 ATP/glucose after accounting for 3 ATP formed by classic substrate-level phosphorylation, and it is one the highest yields for any glucose fermentation. The yield was the same when unsaturated fatty acid bonds, not H+, served as the electron acceptor (as during biohydrogenation. Possession of both Ech and Rnf had been previously documented in only a few sulfate-reducers, was rare in other rumen prokaryotic genomes in our analysis, and may confer an energetic advantage to rumen butyrivibrios. This unique energy conservation system might enhance the butyrivibrios’ ability to overcome growth inhibition by unsaturated fatty acids, as postulated herein.

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

Science.gov (United States)

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

2014-07-01

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

Elevated levels of mitochonrial respiratory complexes activities and ATP production in 17-β-estradiol-induced prolactin-secretory tumor cells in male rats are inhibited by melatonin in vivo and in vitro.

Science.gov (United States)

Wang, Bao-Qiang; Yang, Quan-Hui; Xu, Rong-Kun; Xu, Jian-Ning

2013-01-01

Our earlier studies indicate that melatonin inhibits the proliferation of prolactinoma and induces apoptosis of pituitary prolactin-secreting tumor in rats. Melatonin has also been shown to induce apoptosis and to reduce the production of ATP in breast tumor cells. This study analyzed the levels of the four mitochondrial respiratory complexes and the production of ATP and also the effects of melatonin treatment of prolactinoma. In the in vivo study, mitochondria were harvested from control pituitaries or prolactinoma collected from the pituitaries of melatonin- and 17-β-estradiol (E2)-treated male rats. In the in vitro study, prolactinoma cells mitochondria were harvested. Activities of the four mitochondrial respiratory complexes were assayed using fluorometer. ATP production of prolactinoma cells was estimated using bioluminescent methods. Elevated levels of four mitochondrial respiratory complexes activities and ATP production were recorded in prolactinoma cells. Moreover, in both in vivo and in vitro studies, melatonin inhibited the activities of mitochondrial respiratory complexes and the production of ATP in prolactinoma cells. There is a link between mitochondrial function increase and tumorigenesis. Melatonin induces apoptosis of pituitary prolactin-secreting tumor of rats via the induction of mitochondrial dysfunction and inhibition of energy metabolism.

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

Science.gov (United States)

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

2018-02-01

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

Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases.

Science.gov (United States)

Zhang, Xiang-Feng; Liu, Shuang; Zhou, Yu-Jie; Zhu, Guang-Fa; Foda, Hussein D

2010-04-05

Exposure of adult mice to more than 95% O(2) produces a lethal injury by 72 hours. Nitric oxide synthase (NOS) is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS. One hundred and forty-four osteopontin knock-out (KO) mice and their matched wild type background control (WT) were exposed in sealed cages > 95% oxygen or room air for 24- 72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase (eNOS) and iNOS mRNA in lung tissues at 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry (IHC) was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues. OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85 +/- 0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31 +/- 0.92 in the WT group (P < 0.05). iNOS mRNA (48 hours: 1.04 +/- 0.08 vs. 0.63 +/- 0.09, P < 0.01; 72 hours: 0.89 +/- 0.08 vs. 0.72 +/- 0.09, P < 0.05) and eNOS mRNA (48 hours: 0.62 +/- 0.08 vs. 0.43 +/- 0.09, P < 0.05; 72 hours: 0.67 +/- 0.08 vs. 0.45 +/- 0.09, P < 0.05) expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS (20.54 +/- 3.18 vs. 12.52 +/- 2.46, P < 0.05) and eNOS (19.83 +/- 5.64 vs. 9.45 +/- 3.82, P < 0.05) in lung tissues of OPN KO mice at 72 hours of hyperoxia. OPN can protect against

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

Science.gov (United States)

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

2014-10-28

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

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

Science.gov (United States)

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

2018-02-28

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

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

International Nuclear Information System (INIS)

Milgrom, Elena M.; Milgrom, Yakov M.

2012-01-01

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

Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity

DEFF Research Database (Denmark)

Hempel, Casper; Kohnke, Hannes; Maretty, Lasse

2014-01-01

Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS...... increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion...

Astakine LvAST binds to the β subunit of F1-ATP synthase and likely plays a role in white shrimp Litopeneaus vannamei defense against white spot syndrome virus.

Science.gov (United States)

Liang, Gao-Feng; Liang, Yan; Xue, Qinggang; Lu, Jin-Feng; Cheng, Jun-Jun; Huang, Jie

2015-03-01

Cytokines play a critical role in innate and adaptive immunity. Astakines represent a group of invertebrate cytokines that are related to vertebrate prokineticin and function in promoting hematopoiesis in crustaceans. We have identified an astakine from the white shrimp Litopeneaus vannamei and named it LvAST in a previous research. In the present research, we investigated the interactions among LvAST, the envelope protein VP37 of white spot syndrome virus (i.e., WSSV), and the β subunit of F1-ATP synthase (ATPsyn-β) of the white shrimp (i.e., BP53) using binding assays and co-precipitations. We also examined the effects of LvAST on shrimp susceptibility to WSSV. We found that LvAST and VP37 competitively bound to BP53, but did not bind to each other. Shrimps that had been injected with recombinant LvAST exhibited significantly lower mortality and longer survival time in experimental infections by WSSV. In contrast, shrimps whose LvAST gene expression had been inhibited by RNA interference showed significantly higher WSSV infection intensity and shorter survival time following viral challenges. These results suggested that LvAST and WSSV both likely use ATPsyn-β as a receptor and LvAST plays a role in shrimp defense against WSSV infection. This represented the first research showing the involvement of astakines in host antiviral immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Hiroaki Nagai

2017-10-01

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

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

Science.gov (United States)

Liu, Guo Jun; Bennett, Max R

2003-11-14

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

Cloning and characterization of ATP synthase CF1 α gene from ...

African Journals Online (AJOL)

ajl yemi

2011-12-19

Dec 19, 2011 ... Kohler RH, Horn R, Lossl A (1991). Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene. Mol. Gen. Genet. 227: 369-376. Langmead B, Schatz MC, Lin J, Pop M, Salzberg SL (2009a). Searching for SNPs with cloud computing. Genom.

Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

Science.gov (United States)

Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

2016-03-01

Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

Science.gov (United States)

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

2013-01-01

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

Renal epithelial cells can release ATP by vesicular fusion

Directory of Open Access Journals (Sweden)

Randi G Bjaelde

2013-09-01

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

Effects of long-term inhibition of neuronal nitric oxide synthase on blood pressure and renin release

DEFF Research Database (Denmark)

Ollerstam, A.; Skøtt, O.; Ek, J.

2001-01-01

Nitric oxide (NO) produced by neuronal NO-synthase (nNOS) in macula densa cells may be involved in the control of renin release. 7-Nitro indazole (7-NI) inhibits nNOS, and we investigated the effect of short- (4 days) and long-term (4 weeks) 7-NI treatment on blood pressure (BP), plasma renin...... LS rats (107 +/- 15 vs. 56 +/- 1 mGU mL(-1)). Stimulation of PRC in LS rats was further enhanced by 7-NI after 4 days of treatment, but not affected in rats treated for 4 weeks. This suggests that inhibition of nNOS stimulates renin release but that this stimulatory effect in the long run might...

Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

DEFF Research Database (Denmark)

Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

2015-01-01

The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...

Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

Directory of Open Access Journals (Sweden)

Dong Ju Son

2014-08-01

Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

Science.gov (United States)

Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

2004-01-30

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

Metabolic Trade-offs in Yeast are Caused by F1F0-ATP synthase

DEFF Research Database (Denmark)

Nilsson, Avlant; Nielsen, Jens

2016-01-01

of intermediary metabolism and consequently metabolic trade-offs may take place. One such trade-off, aerobic fermentation, occurs in both yeast (the Crabtree effect) and cancer cells (the Warburg effect) and has been a scientific challenge for decades. Here we show, using flux balance analysis combined...... with in vitro measured enzyme specific activities, that fermentation is more catalytically efficient than respiration, i.e. it produces more ATP per protein mass. And that the switch to fermentation at high growth rates therefore is a consequence of a high ATP production rate, provided by a limited pool...

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

Science.gov (United States)

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

2015-01-01

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

Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach

DEFF Research Database (Denmark)

Krath, Britta N.; Hove-Jensen, Bjarne

1999-01-01

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2...

A genetic polymorphism in the sex-linked ATP5A1 gene is associated with individual fitness in Ovenbirds (Seiurus aurocapilla)

Science.gov (United States)

Judith D. Toms; Lori S. Eggert; Wayne J. Arendt; John Faaborg

2012-01-01

While testing genetic sexing techniques in Ovenbirds (Seiurus aurocapilla),we found a genetic polymorphism in the ATP5A1 gene in 38% of individuals. The Z ' allele included changes in both intronic and exonic portions of the sequenced region, but there was no evidence that this changed the resulting ATP synthase product. Males that had one or more copies of...

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

International Nuclear Information System (INIS)

Kazimov, A.K.

1975-01-01

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

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

Science.gov (United States)

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

Science.gov (United States)

Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

2013-12-01

Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

Target-site resistance to acetolactate synthase (ALS)-inhibiting herbicides in Amaranthus palmeri from Argentina.

Science.gov (United States)

Larran, Alvaro S; Palmieri, Valeria E; Perotti, Valeria E; Lieber, Lucas; Tuesca, Daniel; Permingeat, Hugo R

2017-12-01

Herbicide-resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides (R1 and R2) were found by farmers in two locations in Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. We performed in vivo dose-response assays, and confirmed that both populations had strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared with a susceptible population (S). In vitro ALS activity inhibition tests only indicated considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations producing substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653. This is the first report of als resistance alleles in A. palmeri in Argentina. The data support the involvement of a target-site mechanism of resistance to ALS-inhibiting herbicides. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Thromboxane synthase suppression induces lung cancer cell apoptosis via inhibiting NF-κB

International Nuclear Information System (INIS)

Leung, Kin Chung; Li, Ming-Yue; Leung, Billy C.S.; Hsin, Michael K.Y.; Mok, Tony S.K.; Underwood, Malcolm J.; Chen, George G.

2010-01-01

Accumulating evidence shows that the inhibition of thromboxane synthase (TXS) induced apoptosis in cancer cells. TXS inhibitor 1-Benzylimidzole (1-BI) can trigger apoptosis in lung cancer cells but the mechanism is not fully defined. In this study, lung cancer cells were treated with 1-BI. In this study, the level of reactive oxygen species (ROS) was measured and NF-κB activity was determined in human lung cancer cells. The roles of ROS and NF-κB in 1-BI-mediated cell death were analyzed. The results showed that 1-BI induced ROS generation but decreased the activity of NF-κB by reducing phosphorylated IκBα (p-IκBα) and inhibiting the translocation of p65 into the nucleus. In contrast to 1-BI, antioxidant N-acetyl cysteine (NAC) stimulated cell proliferation and significantly protected the cells from 1-BI-mediated cell death by neutralizing ROS. Collectively, apoptosis induced by 1-BI is associated with the over-production of ROS and the reduction of NF-κB. Antioxidants can significantly block the inhibitory effect of 1-BI.

Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.

Science.gov (United States)

Boutsalis, P; Powles, S B

1995-07-01

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that Km and Vmax did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F1, F2 and F3 generations. F1 hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F2 generation following chlorsulfuron application. A segregation ratio of 1∶2∶1 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F3 families, derived from intermediate F2 individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS.

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

Science.gov (United States)

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

2016-06-01

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

Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity.

Science.gov (United States)

Schirris, Tom J J; Ritschel, Tina; Herma Renkema, G; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

2015-09-29

Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

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

Science.gov (United States)

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

2005-01-01

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

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

Science.gov (United States)

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

2017-12-29

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

Eckmaxol, a Phlorotannin Extracted from Ecklonia maxima, Produces Anti-β-amyloid Oligomer Neuroprotective Effects Possibly via Directly Acting on Glycogen Synthase Kinase 3β.

Science.gov (United States)

Wang, Jialing; Zheng, Jiachen; Huang, Chunhui; Zhao, Jiaying; Lin, Jiajia; Zhou, Xuezhen; Naman, C Benjamin; Wang, Ning; Gerwick, William H; Wang, Qinwen; Yan, Xiaojun; Cui, Wei; He, Shan

2018-04-10

Alzheimer's disease is a progressive neurodegenerative disorder that mainly affects the elderly. Soluble β-amyloid oligomer, which can induce neurotoxicity, is generally regarded as the main neurotoxin in Alzheimer's disease. Here we report that eckmaxol, a phlorotannin extracted from the brown alga Ecklonia maxima, could produce neuroprotective effects in SH-SY5Y cells. Eckmaxol effectively prevented but did not rescue β-amyloid oligomer-induced neuronal apoptosis and increase of intracellular reactive oxygen species. Eckmaxol also significantly reversed the decreased expression of phospho-Ser9-glycogen synthase kinase 3β and increased expression of phospho-extracellular signal-regulated kinase, which was induced by Aβ oligomer. Moreover, both glycogen synthase kinase 3β and mitogen activated protein kinase inhibitors produced neuroprotective effects in SH-SY5Y cells. Furthermore, eckmaxol showed favorable interaction in the ATP binding site of glycogen synthase kinase 3β and mitogen activated protein kinase. These results suggested that eckmaxol might produce neuroprotective effects via concurrent inhibition of glycogen synthase kinase 3β and extracellular signal-regulated kinase pathways, possibly via directly acting on glycogen synthase kinase 3β and mitogen activated protein kinase. Based on the central role that β-amyloid oligomers play in the pathogenesis of Alzheimer's disease and the high annual production of Ecklonia maxima for alginate and other nutritional ingredients, this report represents a new candidate for the treatment of Alzheimer's disease, and also expands the potential application of Ecklonia maxima and its constituents in the field of pharmacology.

Up-regulation of insulin-like growth factor 2 by ketamine requires glycogen synthase kinase-3 inhibition

Science.gov (United States)

Grieco, Steven F.; Cheng, Yuyan; Eldar-Finkelman, Hagit; Jope, Richard S.; Beurel, Eléonore

2016-01-01

An antidepressant dose of the rapidly-acting ketamine inhibits glycogen synthase kinase-3 (GSK3) in mouse hippocampus, and this inhibition is required for the antidepressant effect of ketamine in learned helplessness depression-like behavior. Here we report that treatment with an antidepressant dose of ketamine (10 mg/kg) increased expression of insulin-like growth factor 2 (IGF2) in mouse hippocampus, an effect that required ketamine-induced inhibition of GSK3. Ketamine also inhibited hippocampal GSK3 and increased expression of hippocampal IGF2 in mice when administered after the induction of learned helplessness. Treatment with the specific GSK3 inhibitor L803-mts was sufficient to up-regulate hippocampal IGF2 expression. Administration of IGF2 siRNA reduced ketamine's antidepressant effect in the learned helplessness paradigm. Mice subjected to the learned helplessness paradigm were separated into two groups, those that were resilient (non-depressed) and those that were susceptible (depressed). Non-depressed resilient mice displayed higher expression of IGF2 than susceptible mice. These results indicate that IGF2 contributes to ketamine's antidepressant effect and that IGF2 may confer resilience to depression-like behavior. PMID:27542584

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

Science.gov (United States)

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

2013-10-01

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

Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

Directory of Open Access Journals (Sweden)

Smith Milton

2006-11-01

Full Text Available Abstract Background 2-Chloroethyl ethyl sulphide (CEES is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD. Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA or dichlorofluorescin diacetate (DCFH-DA. Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS

Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

International Nuclear Information System (INIS)

Miziorko, H.M.; Behnke, C.E.

1985-01-01

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.

Science.gov (United States)

McEachern, Kerry Anne; Fung, John; Komarnitsky, Svetlana; Siegel, Craig S; Chuang, Wei-Lien; Hutto, Elizabeth; Shayman, James A; Grabowski, Gregory A; Aerts, Johannes M F G; Cheng, Seng H; Copeland, Diane P; Marshall, John

2007-07-01

An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease (D409V/null). Biochemical characterization of Genz-112638 showed good potency (IC(50) approximately 24nM) and specificity against the target enzyme. Mice that received drug prior to significant accumulation of substrate (10 weeks of age) showed reduced levels of glucosylceramide and number of Gaucher cells in the spleen, lung and liver when compared to age-matched control animals. Treatment of older mice that already displayed significant amounts of tissue glucosylceramide (7 months old) resulted in arrest of further accumulation of the substrate and appearance of additional Gaucher cells in affected organs. These data indicate that substrate inhibition therapy with Genz-112638 represents a viable alternate approach to enzyme therapy to treat the visceral pathology in Gaucher disease.

The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Gnipová, Anna; Šubrtová, Karolína; Panicucci, Brian; Horváth, A.; Lukeš, Julius; Zíková, Alena

2015-01-01

Roč. 14, č. 3 (2015), s. 297-310 ISSN 1535-9778 R&D Projects: GA MŠk LL1205; GA ČR GAP302/12/2513 EU Projects: European Commission(XE) 316304 Institutional support: RVO:60077344 Keywords : cytochrome c-oxidase * structural basis * mitochondrial ATP synthase Subject RIV: EE - Microbiology, Virology Impact factor: 2.946, year: 2015

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

Science.gov (United States)

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

1989-07-01

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

Inhibition of glycogen synthase kinase-3β attenuates glucocorticoid-induced suppression of myogenic differentiation in vitro.

Directory of Open Access Journals (Sweden)

Zhenyu Ma

Full Text Available Glucocorticoids are the only therapy that has been demonstrated to alter the progress of Duchenne muscular dystrophy (DMD, the most common muscular dystrophy in children. However, glucocorticoids disturb skeletal muscle metabolism and hamper myogenesis and muscle regeneration. The mechanisms involved in the glucocorticoid-mediated suppression of myogenic differentiation are not fully understood. Glycogen synthase kinase-3β (GSK-3β is considered to play a central role as a negative regulator in myogenic differentiation. Here, we showed that glucocorticoid treatment during the first 48 h in differentiation medium decreased the level of phosphorylated Ser9-GSK-3β, an inactive form of GSK-3β, suggesting that glucocorticoids affect GSK-3β activity. We then investigated whether GSK-3β inhibition could regulate glucocorticoid-mediated suppression of myogenic differentiation in vitro. Two methods were employed to inhibit GSK-3β: pharmacological inhibition with LiCl and GSK-3β gene knockdown. We found that both methods resulted in enhanced myotube formation and increased levels of muscle regulatory factors and muscle-specific protein expression. Importantly, GSK-3β inhibition attenuated glucocorticoid-induced suppression of myogenic differentiation. Collectively, these data suggest the involvement of GSK-3β in the glucocorticoid-mediated impairment of myogenic differentiation. Therefore, the inhibition of GSK-3β may be a strategy for preventing glucocorticoid-induced muscle degeneration.

Changes in H(+)-ATP Synthase Activity, Proton Electrochemical Gradient, and pH in Pea Chloroplast Can Be Connected with Variation Potential.

Science.gov (United States)

Sukhov, Vladimir; Surova, Lyubov; Morozova, Ekaterina; Sherstneva, Oksana; Vodeneev, Vladimir

2016-01-01

Local stimulation induces generation and propagation of electrical signals, including the variation potential (VP) and action potential, in plants. Burning-induced VP changes the physiological state of plants; specifically, it inactivates photosynthesis. However, the mechanisms that decrease photosynthesis are poorly understood. We investigated these mechanisms by measuring VP-connected systemic changes in CO2 assimilation, parameters of light reactions of photosynthesis, electrochromic pigment absorbance shifts, and light scattering. We reveal that inactivation of photosynthesis in the pea, including inactivation of dark and light reactions, was connected with the VP. Inactivation of dark reactions decreased the rate constant of the fast relaxation of the electrochromic pigment absorbance shift, which reflected a decrease in the H(+)-ATP synthase activity. This decrease likely contributed to the acidification of the chloroplast lumen, which developed after VP induction. However, VP-connected decrease of the proton motive force across the thylakoid membrane, possibly, reflected a decreased pH in the stroma. This decrease may be another mechanism of chloroplast lumen acidification. Overall, stroma acidification can decrease electron flow through photosystem I, and lumen acidification induces growth of fluorescence non-photochemical quenching and decreases electron flow through photosystem II, i.e., pH decreases in the stroma and lumen, possibly, contribute to the VP-induced inactivation of light reactions of photosynthesis.

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

Science.gov (United States)

Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

2012-05-01

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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

Directory of Open Access Journals (Sweden)

Benjamin Clémençon

2012-02-01

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

The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

Science.gov (United States)

Hall, Justin; Ralph, Erik C; Shanker, Suman; Wang, Hong; Byrnes, Laura J; Horst, Reto; Wong, Jimson; Brault, Amy; Dumlao, Darren; Smith, James F; Dakin, Leslie A; Schmitt, Daniel C; Trujillo, John; Vincent, Fabien; Griffor, Matt; Aulabaugh, Ann E

2017-12-01

Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2',3'-cGAMP formation, and interestingly, describe a catalytic mechanism where 2',3'-cGAMP may be a minor product of cGAS compared with linear nucleotides. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

St36 electroacupuncture activates nNOS, iNOS and ATP-sensitive potassium channels to promote orofacial antinociception in rats.

Science.gov (United States)

Almeida, R T; Galdino, G; Perez, A C; Silva, G; Romero, T R; Duarte, I D

2017-02-01

Orofacial pain is pain perceived in the face and/or oral cavity, generally caused by diseases or disorders of regional structures, by dysfunction of the nervous system, or through referral from distant sources. Treatment of orofacial pain is mainly pharmacological, but it has increased the number of reports demonstrating great clinical results with the use of non-pharmacological therapies, among them electroacupuncture. However, the mechanisms involved in the electroacupuncture are not well elucidated. Thus, the present study investigate the involvement of the nitric oxide synthase (NOS) and ATP sensitive K + channels (KATP) in the antinociception induced by electroacupuncture (EA) at acupoint St36. Thermal nociception was applied in the vibrissae region of rats, and latency time for face withdrawal was measured. Electrical stimulation of acupoint St36 for 20 minutes reversed the thermal withdrawal latency and this effect was maintained for 150 min. Intraperitoneal administration of specific inhibitors of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and a KATP channels blocker reversed the antinociception induced by EA. Furthermore, nitrite concentration in cerebrospinal fluid (CSF) and plasma, increased 4 and 3-fold higher, respectively, after EA. This study suggests that NO participates of antinociception induced by EA by nNOS, iNOS and ATP-sensitive K + channels activation.

The diarylquinoline TMC207 for multidrug-resistant tuberculosis

NARCIS (Netherlands)

Diacon, Andreas H.; Pym, Alexander; Grobusch, Martin; Patientia, Ramonde; Rustomjee, Roxana; Page-Shipp, Liesl; Pistorius, Christoffel; Krause, Rene; Bogoshi, Mampedi; Churchyard, Gavin; Venter, Amour; Allen, Jenny; Palomino, Juan Carlos; de Marez, Tine; van Heeswijk, Rolf P. G.; Lounis, Nacer; Meyvisch, Paul; Verbeeck, Johan; Parys, Wim; de Beule, Karel; Andries, Koen; Mc Neeley, David F.

2009-01-01

BACKGROUND: The diarylquinoline TMC207 offers a new mechanism of antituberculosis action by inhibiting mycobacterial ATP synthase. TMC207 potently inhibits drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro and shows bactericidal activity in patients who have drug-susceptible

Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

Institute of Scientific and Technical Information of China (English)

Shengqiang Chen; Xuegang Luo; Quan Yang; Weiwen Sun; Kaiyi Cao; Xi Chen; Yueling Huang; Lijun Dai; Yonghong Yi

2011-01-01

In the present study, Fmr1 knockout mice (KO mice) were used as the model for fragile X syndrome. The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts, indicating a learning and memory disorder. After treatment with 30, 60, 90, 120, or 200 mg/kg lithium chloride, the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated, in particular, the 200 mg/kg lithium chloride treatment had the most significant effect. Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta, an inactive form of glycogen synthase kinase 3 beta, in the cerebral cortex and hippocampus of the Fmr1 KO mice. These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice, possibly by inhibiting glycogen synthase kinase 3 beta activity.

Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

Science.gov (United States)

Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J; Stunden, H James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R; Cain, Jason E; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P

2017-10-03

Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans. Copyright © 2017 Pépin et al.

Local inhibition of hippocampal nitric oxide synthase does not impair place learning in the Morris water escape task in rats.

Science.gov (United States)

Blokland, A; de Vente, J; Prickaerts, J; Honig, W; Markerink-van Ittersum, M; Steinbusch, H

1999-01-01

Recent studies have provided evidence that nitric oxide (NO) has a role in certain forms of memory formation. Spatial learning is one of the cognitive abilities that has been found to be impaired after systemic administration of an NO-synthase inhibitor. As the hippocampus has a pivotal role in spatial orientation, the present study examined the role of hippocampal NO in spatial learning and reversal learning in a Morris task in adult rats. It was found that N omega-nitro-L-arginine infusions into the dorsal hippocampus affected the manner in which the rats were searching the submerged platform during training, but did not affect the efficiency to find the spatial location of the escape platform. Hippocampal NO-synthase inhibition did not affect the learning of a new platform position in the same water tank (i.e. reversal learning). Moreover, no treatment effects were observed in the probe trials (i.e. after acquisition and after reversal learning), indicating that the rats treated with N omega-nitro-L-arginine had learned the spatial location of the platform. These findings were obtained under conditions where the NO synthesis in the dorsal hippocampus was completely inhibited. On the basis of the present data it was concluded that hippocampal NO is not critically involved in place learning in rats.

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

Directory of Open Access Journals (Sweden)

Helena H. Chowdhury

2014-10-01

Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

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

Directory of Open Access Journals (Sweden)

Ticozzi Paolo

2005-07-01

Full Text Available Abstract Background We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. Results Local, oral or intravenous administration of a single dose of oATP significantly reduced thermal hyperalgesia in hind paws of rats for 24 hours, and such effect was greater than that induced by diclofenac or indomethacin. Following oATP treatment, the expression of the pro-inflammatory chemokines interferon-gamma-inducible protein-10 (IP-10, mon ocyte chemoattractant protein-1 (MCP-1 and interleukin-8 (IL-8 within the inflamed tissues markedly decreased on vessels and infiltrated cells. In parallel, the immunohistochemical findings showed an impairment, with respect to the untreated rats, in P2X7 expression, mainly on nerves and vessels close to the site of inflammation. Finally, oATP treatment significantly reduced the presence of infiltrating inflammatory macrophages in the paw tissue. Conclusion Taken together these results clearly show that oATP reduces carrageenan-induced inflammation in rats.

Carbonic anhydrase activity of integral-functional complexes of thylakoid membranes of spinach chloroplasts

Directory of Open Access Journals (Sweden)

A. V. Semenihin

2015-06-01

Full Text Available Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1 within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1. Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

International Nuclear Information System (INIS)

Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

2005-01-01

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion

Propolis attenuates oxidative injury in brain and lung of nitric oxide synthase inhibited rats

Directory of Open Access Journals (Sweden)

Zeliha Selamoglu-Talas

2015-10-01

Full Text Available Background: The blocking of nitric oxide synthase (NOS activity may reason vasoconstriction with formation of reactive oxygen species. Propolis has biological and pharmacological properties, such as antioxidant. The aim of this study was to examine the antioxidant effects of propolis which natural product on biochemical parameters in brain and lung tissues of acute nitric oxide synthase inhibited rats by Nω-nitro-L-arginine methyl ester (L-NAME.Methods: Rats have been received L-NAME (40 mg/kg, intraperitoneally, NOS inhibitor for 15 days to produce hypertension and propolis (200mg/kg, by gavage the lastest 5 of 15 days.Results: There  were  the  increase  (P<0.001  in  the  malondialdehyde  levels  in  the  L-NAME treatment groups when compared to control rats, but the decrease (P<0.001 in the catalase activities in both brain and lung tissues. There were statistically changes (P<0.001 in these parameters of L-NAME+propolis treated rats as compared with L-NAME-treated group.Conclusion: The application of L-NAME to the Wistar rats resulted in well developed oxidative stress. Also, propolis may influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of hypertensive diseases and oxidative stress.

Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

Directory of Open Access Journals (Sweden)

Carmen Gherasim

Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

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.

Gemfibrozil, a Lipid-lowering Drug, Inhibits the Induction of Nitric-oxide Synthase in Human Astrocytes*

Science.gov (United States)

Pahan, Kalipada; Jana, Malabendu; Liu, Xiaojuan; Taylor, Bradley S.; Wood, Charles; Fischer, Susan M.

2007-01-01

Gemfibrozil, a lipid-lowering drug, inhibited cytokine-induced production of NO and the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astrocytes. Similar to gemfibrozil, clofibrate, another fibrate drug, also inhibited the expression of iNOS. Inhibition of human iNOS promoter-driven luciferase activity by gemfibrozil in cytokine-stimulated U373MG astroglial cells suggests that this compound inhibits the transcription of iNOS. Since gemfibrozil is known to activate peroxisome proliferator-activated receptor-α (PPAR-α), we investigated the role of PPAR-α in gemfibrozil-mediated inhibition of iNOS. Gemfibrozil induced peroxisome proliferator-responsive element (PPRE)-dependent luciferase activity, which was inhibited by the expression of ΔhPPAR-α, the dominant-negative mutant of human PPAR-α. However, ΔhPPAR-α was unable to abrogate gemfibrozil-mediated inhibition of iNOS suggesting that gemfibrozil inhibits iNOS independent of PPAR-α. The human iNOS promoter contains consensus sequences for the binding of transcription factors, including interferon-γ (IFN-γ) regulatory factor-1 (IRF-1) binding to interferon-stimulated responsive element (ISRE), signal transducer and activator of transcription (STAT) binding to γ-activation site (GAS), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein β (C/EBPβ); therefore, we investigated the effect of gemfibrozil on the activation of these transcription factors. The combination of interleukin (IL)-1β and IFN-γ induced the activation of NF-κB, AP-1, C/EBPβ, and GAS but not that of ISRE, suggesting that IRF-1 may not be involved in cytokine-induced expression of iNOS in human astrocytes. Interestingly, gemfibrozil strongly inhibited the activation of NF-κB, AP-1, and C/EBPβ but not that of GAS in cytokine-stimulated astroglial cells. These results suggest that gemfibrozil inhibits the induction of iNOS probably by

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

International Nuclear Information System (INIS)

Mithieux, G.; Rousset, B.

1989-01-01

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

Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

Science.gov (United States)

2011-01-01

Background Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. Results NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. Conclusion Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses. PMID:22035583

Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3.

Science.gov (United States)

Feyt, Christine; Kienlen-Campard, Pascal; Leroy, Karelle; N'Kuli, Francisca; Courtoy, Pierre J; Brion, Jean-Pierre; Octave, Jean-Noël

2005-09-30

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells.

Science.gov (United States)

Zhou, Weibo; Simpson, P Jeanette; McFadden, Jill M; Townsend, Craig A; Medghalchi, Susan M; Vadlamudi, Aravinda; Pinn, Michael L; Ronnett, Gabriele V; Kuhajda, Francis P

2003-11-01

C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the

Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

Directory of Open Access Journals (Sweden)

Quadros Edward V

2009-05-01

Full Text Available Abstract Background Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration. Methods Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM, or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2. Results There was a high correlation of cell growth with ATP concentration (r = 0.948 in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p Conclusion Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies.

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

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...... of levcromakalim (0.025 mg kg(-1)), pinacidil (0.38 mg kg(-1)) and P-1075 (0.016 mg kg(-1)) in rat MMA. In vitro PNU-37883A significantly inhibited the dilatory responses of the three K-ATP channel openers in rat MMA at 10(-7) and 3 x 10(-7) M. Conclusions and implications: We suggest that Kir6.1/SUR2B...

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

International Nuclear Information System (INIS)

Gunter, Thomas E.; Gerstner, Brent; Lester, Tobias; Wojtovich, Andrew P.; Malecki, Jon; Swarts, Steven G.; Brookes, Paul S.; Gavin, Claire E.; Gunter, Karlene K.

2010-01-01

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn 2+ inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn 2+ inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn 2+ inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F 1 F 0 ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn 2+ inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Cutting edge: Antimalarial drugs inhibit IFN-β production through blockade of cyclic GMP-AMP synthase-DNA interaction.

Science.gov (United States)

An, Jie; Woodward, Joshua J; Sasaki, Tomikazu; Minie, Mark; Elkon, Keith B

2015-05-01

Type I IFN is strongly implicated in the pathogenesis of systemic autoimmune diseases, such as lupus, and rare monogenic IFNopathies, including Aicardi-Goutières syndrome. Recently, a new DNA-activated pathway involving the enzyme cyclic GMP-AMP synthase (cGAS) was described and potentially linked to Aicardi-Goutières syndrome. To identify drugs that could potentially inhibit cGAS activity, we performed in silico screening of drug libraries. By computational analysis, we identified several antimalarial drugs (AMDs) that were predicted to interact with the cGAS/dsDNA complex. Our studies validated that several AMDs were effective inhibitors of IFN-β production and that they functioned by inhibiting dsDNA stimulation of cGAS. Because AMDs have been widely used in human diseases and have an excellent safety profile, our findings suggest new therapeutic strategies for the treatment of severe debilitating diseases associated with type I IFNs due to cGAS activation. Copyright © 2015 by The American Association of Immunologists, Inc.

Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.

Science.gov (United States)

Yutani, Masahiro; Hashimoto, Yukie; Ogita, Akira; Kubo, Isao; Tanaka, Toshio; Fujita, Ken-ichi

2011-11-01

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well-known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the baker's yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625 mM. A hyperosmotic condition (1.2 M sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose-dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition. Copyright © 2011 John Wiley & Sons, Ltd.

Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

DEFF Research Database (Denmark)

Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

2000-01-01

A steady state kinetic investigation of the Pi activation of 5-phospho-D-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased...... the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas...... saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Mahalingan, Krishna K.; Baskaran†, Sulochanadevi; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D. (Indiana-Med)

2017-01-10

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

Science.gov (United States)

Michel, J B; Feron, O; Sase, K; Prabhakar, P; Michel, T

1997-10-10

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Motor pathway excitability in ATP13A2 mutation carriers

DEFF Research Database (Denmark)

Zittel, S; Kroeger, J; van der Vegt, J P M

2012-01-01

OBJECTIVE: To describe excitability of motor pathways in Kufor-Rakeb syndrome (PARK9), an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration caused by a mutation in the ATP13A2 gene, using transcranial magnetic stimulation (TMS). METHODS: Five members of a Chilean family...... with an ATP13A2 mutation (one affected mutation carrier (MC) with a compound heterozygous mutation, 4 asymptomatic MC with a single heterozygous mutation) and 11 healthy subjects without mutations were studied. We measured motor evoked potentials (MEP), the contralateral silent period (cSP), short interval....... RESULTS: CSP duration was increased in the symptomatic ATP13A2 MC. The iSP measurements revealed increased interhemispheric inhibition in both the compound heterozygous and the heterozygous MC. CONCLUSION: A compound heterozygous mutation in the ATP13A2 gene is associated with increased intracortical...

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Upregulation of cyclooxygenase-2 expression in porcine macula densa with chronic nitric oxide synthase inhibition.

Science.gov (United States)

Kommareddy, M; McAllister, R M; Ganjam, V K; Turk, J R; Laughlin, M Harold

2011-11-01

The objective of this study was to investigate the effects of chronic inhibition of nitric oxide synthase (NOS) on cyclooxygenase-2 (COX-2) expression in the macula densa (MD) of swine, as well as the effects on expression of related proteins. Adult female Yucatan swine were given either tap water (control, n = 6) or water with N (G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/liter, n = 5) for a minimum of 30 days. Duplicate samples of kidney were fixed or snap frozen. There was a significant (P = .0082) upregulation of COX-2 mRNA expression in the MD of L-NAME, as well as an apparent increase in COX-2 protein. Plasma renin activity also increased with L-NAME treatment (control, 0.34 ± 0.08 ng/ml; L-NAME, 1.26 ± 0.03 ng/ml; P = .00000003). There were no differences between groups in expression of either inducible NOS or renin protein or in serum electrolyte concentrations. In conclusion, with chronic inhibition of NOS, COX-2 in MD is upregulated, perhaps to compensate for loss of nitric oxide. Increases in COX-2 products may counteract renal arteriolar constriction and sustain renin release.

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

Science.gov (United States)

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

2011-11-01

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

The Methanolic Extract from Murraya koenigii L. Inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+ Channel as Antinociceptive Mechanism

Directory of Open Access Journals (Sweden)

Nushrat Sharmin Ani

2016-01-01

Full Text Available Murraya koenigii L. is a perennial shrub, belonging to the family Rutaceae. Traditionally, the leaves of this plant are extensively used in treatment of a wide range of diseases and disorders including pain and inflammation. Although researchers have revealed the antinociceptive effects of this plant’s leaves during past few years, the mechanisms underlying these effects are still unknown. Therefore, the present study evaluated some antinociceptive mechanisms of the methanolic extract of M. koenigii (MEMK leaves along with its antinociceptive potential using several animal models. The antinociceptive effects of MEMK were evaluated using formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200 mg/kg. In addition, we also justified the possible participations of glutamatergic system and ATP-sensitive potassium channels in the observed activities. Our results demonstrated that MEMK significantly (p<0.01 inhibited the pain thresholds induced by formalin and acetic acid in a dose-dependent manner. MEMK also significantly (p<0.01 suppressed glutamate-induced pain. Moreover, pretreatment with glibenclamide (an ATP-sensitive potassium channel blocker at 10 mg/kg significantly (p<0.05 reversed the MEMK-mediated antinociception. These revealed that MEMK might have the potential to interact with glutamatergic system and the ATP-sensitive potassium channels to exhibit its antinociceptive activities. Therefore, our results strongly support the antinociceptive effects of M. koenigii leaves and provide scientific basis of their analgesic uses in the traditional medicine.

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

Inhibition of hematopoietic prostaglandin D2 synthase (H-PGDS) by an alkaloid extract from Combretum molle.

Science.gov (United States)

Moyo, Rejoice; Chimponda, Theresa; Mukanganyama, Stanley

2014-07-05

Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD2. Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated. H-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates. A specific activity of 24 μmol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 μg/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with Ki = 41 μg/ml towards GSH, and non-competitive inhibition towards CDNB with Ki = 7.7 μg/ml and Ki' = 9.2 μg/ml. The data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation.

Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

Energy Technology Data Exchange (ETDEWEB)

Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

2014-03-01

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

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

Science.gov (United States)

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

2009-09-01

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

Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

Energy Technology Data Exchange (ETDEWEB)

Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)

2014-11-01

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

Comparative analysis of miRNAs of two rapeseed genotypes in response to acetohydroxyacid synthase-inhibiting herbicides by high-throughput sequencing.

Directory of Open Access Journals (Sweden)

Maolong Hu

Full Text Available Acetohydroxyacid synthase (AHAS, also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI, sulfonylureas (SU, pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs after SU herbicide application than in sensitive genotype N131 (164 miRNAs. In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

Science.gov (United States)

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycogen Synthase Kinase 3β Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

Directory of Open Access Journals (Sweden)

Liang Ma

2013-08-01

Full Text Available Alternative strategies beyond current chemotherapy and radiation therapy regimens are needed in the treatment of advanced stage and recurrent endometrial cancers. There is considerable promise for biologic agents targeting the extracellular signal-regulated kinase (ERK pathway for treatment of these cancers. Many downstream substrates of the ERK signaling pathway, such as glycogen synthase kinase 3β (GSK3β, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3β inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3β by either lithium chloride (LiCl or specific GSK3β inhibitor VIII showed cytostatic and cytotoxic effects on multiple endometrial cancer cell lines, with little effect on the immortalized normal endometrial cell line. Flow cytometry and immunofluorescence revealed a G2/M cell cycle arrest in both type I (AN3CA, KLE, and RL952 and type II (ARK1 endometrial cancer cell lines. In addition, LiCl pre-treatment sensitized AN3CA cells to the chemotherapy agent paclitaxel. Administration of LiCl to AN3CA tumor-bearing mice resulted in partial or complete regression of some tumors. Thus, GSK3β activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

2015-03-13

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

International Nuclear Information System (INIS)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

2015-01-01

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

Effect of systemic nitric oxide synthase inhibition on optic disc oxygen partial pressure in normoxia and in hypercapnia.

Science.gov (United States)

Petropoulos, Ioannis K; Pournaras, Jean-Antoine C; Stangos, Alexandros N; Pournaras, Constantin J

2009-01-01

To investigate the effect of systemic nitric oxide synthase (NOS) inhibition on optic disc oxygen partial pressure (PO(2)) in normoxia and hypercapnia. Intervascular optic disc PO(2) was measured in 12 anesthetized minipigs by using oxygen-sensitive microelectrodes placed 0.1), despite a 21% increase of mean arterial pressure. Optic disc PO(2) increase under hypercapnia was blunted after L-NAME injection (DeltaPO(2) = 0.6 +/- 1.1 mm Hg; 3%; P > 0.1), and this effect was reversible by L-arginine. Moreover, L-NAME reduced the response to carbogen by 29% (DeltaPO(2) = 9.1 +/- 4.4 mm Hg; 49%; P = 0.01 versus before L-NAME). The response to hyperoxia was not affected. Whereas systemic NOS inhibition did not affect optic disc PO(2) in normoxia, a blunting effect was noted on the CO(2)-induced optic disc PO(2) increase. Nitric oxide appears to mediate the hypercapnic optic disc PO(2) increase.

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-09-09

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

Directory of Open Access Journals (Sweden)

Р. В. Фафула

2017-12-01

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

Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

Directory of Open Access Journals (Sweden)

Lu Frances Fangjia

2012-11-01

Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

Hypo-and hyperthyroidism affect the ATP, ADP and AMP hydrolysis in rat hippocampal and cortical slices.

Science.gov (United States)

Bruno, Alessandra Nejar; Diniz, Gabriela Placoná; Ricachenevsky, Felipe Klein; Pochmann, Daniela; Bonan, Carla Denise; Barreto-Chaves, Maria Luiza M; Sarkis, João José Freitas

2005-05-01

The presence of severe neurological symptoms in thyroid diseases has highlighted the importance of thyroid hormones in the normal functioning of the mature brain. Since, ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system (CNS), the ectonucleotidase cascade that hydrolyzes ATP to adenosine, is also involved in the control of brain functions. Thus, we investigated the influence of hyper-and hypothyroidism on the ATP, ADP and AMP hydrolysis in hippocampal and cortical slices from adult rats. Hyperthyroidism was induced by daily injections of l-thyroxine (T4) 25 microg/100 g body weight, for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Hypothyroid rats were hormonally replaced by daily injections of T4 (5 microg/100 g body weight, i.p.) for 5 days. Hyperthyroidism significantly inhibited the ATP, ADP and AMP hydrolysis in hippocampal slices. In brain cortical slices, hyperthyroidism inhibited the AMP hydrolysis. In contrast, hypothyroidism increased the ATP, ADP and AMP hydrolysis in both hippocampal and cortical slices and these effects were reverted by T4 replacement. Furthermore, hypothyroidism increased the expression of NTPDase1 and 5'-nucleotidase, whereas hyperthyroidism decreased the expression of 5'-nucleotidase in hippocampus of adult rats. These findings demonstrate that thyroid disorders may influence the enzymes involved in the complete degradation of ATP to adenosine and possibly affects the responses mediated by adenine nucleotides in the CNS of adult rats.

Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

Energy Technology Data Exchange (ETDEWEB)

Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH

2012-04-24

Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

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

Science.gov (United States)

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

2017-01-01

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

Nisin depletes ATP and proton motive force in mycobacteria.

Science.gov (United States)

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

2000-12-01

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

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

Directory of Open Access Journals (Sweden)

Hsu Che-Fang

2009-09-01

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

Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.

Science.gov (United States)

Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong

2014-05-01

mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.

Wild-type phosphoribosylpyrophosphate synthase (PRS from Mycobacterium tuberculosis: a bacterial class II PRS?

Directory of Open Access Journals (Sweden)

Ardala Breda

Full Text Available The 5-phospho-α-D-ribose 1-diphosphate (PRPP metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS. Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure

Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

Science.gov (United States)

Breda, Ardala; Martinelli, Leonardo K. B.; Bizarro, Cristiano V.; Rosado, Leonardo A.; Borges, Caroline B.; Santos, Diógenes S.; Basso, Luiz A.

2012-01-01

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of Mt

Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum.

Science.gov (United States)

Melani, Alessia; Corti, Francesca; Stephan, Holger; Müller, Christa E; Donati, Chiara; Bruni, Paola; Vannucchi, Maria Giuliana; Pedata, Felicita

2012-01-01

In the central nervous system (CNS) ATP and adenosine act as transmitters and neuromodulators on their own receptors but it is still unknown which part of extracellular adenosine derives per se from cells and which part is formed from the hydrolysis of released ATP. In this study extracellular concentrations of adenosine and ATP from the rat striatum were estimated by the microdialysis technique under in vivo physiological conditions and after focal ischemia induced by medial cerebral artery occlusion. Under physiological conditions, adenosine and ATP concentrations were in the range of 130 nmol/L and 40 nmol/L, respectively. In the presence of the novel ecto-ATPase inhibitor, PV4 (100 nmol/L), the extracellular concentration of ATP increased 12-fold to ~360 nmol/L but the adenosine concentration was not altered. This demonstrates that, under physiological conditions, adenosine is not a product of extracellular ATP. In the first 4h after ischemia, adenosine increased to ~690 nmol/L and ATP to ~50 nmol/L. In the presence of PV4 the extracellular concentration of ATP was in the range of 450 nmol/L and a significant decrease in extracellular adenosine (to ~270 nmol/L) was measured. The contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia onset. Furthermore we demonstrated, by immunoelectron microscopy, the presence of the concentrative nucleoside transporter CNT2 on plasma and vesicle membranes isolated from the rat striatum. These results are in favor that adenosine is transported in vesicles and is released in an excitation-secretion manner under in vivo physiological conditions. Early after ischemia, extracellular ATP is hydrolyzed by ecto-nucleotidases which significantly contribute to the increase in extracellular adenosine. To establish the contribution of extracellular ATP to adenosine might constitute the basis for devising a correct putative purinergic strategy aimed at protection from ischemic damage

The P2X7 ATP receptor modulates renal cyst development in vitro

International Nuclear Information System (INIS)

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

2004-01-01

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

ATP Release Channels

Directory of Open Access Journals (Sweden)

Akiyuki Taruno

2018-03-01

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

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.

Science.gov (United States)

Sakato, Miho; King, Stephen M

2003-10-31

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.

Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

Science.gov (United States)

Lee, Ming Hong; Kim, Jae Yeon; Yoon, Jeong Hoon; Lim, Hyo Jin; Kim, Tae Hee; Jin, Changbae; Kwak, Wie-Jong; Han, Chang-Kyun; Ryu, Jae-Ha

2006-09-01

Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

Science.gov (United States)

Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

2004-11-15

Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

DEFF Research Database (Denmark)

Christensen, Caspar Elo; Kragelund, Birthe B; von Wettstein-Knowles, Penny

2007-01-01

Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

Lithium Impairs Kidney Development and Inhibits Glycogen Synthase Kinase-3β in Collecting Duct Principal Cells

DEFF Research Database (Denmark)

Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

level significantly whereas total GSK-3β abundance was unaltered. Li+ treatment increased α-Smooth Muscle Actin (α-SMA) protein level significantly whereas E-cadherin expression was unaltered. In summary, Li+ treatment impairs postnatal development of the kidney cortex and outer medulla and increases pGSK......The postnatal rat kidney is highly susceptible to Lithium (Li+), which leads to significant tissue injury. We hypothesized that Li+ impairs development of the kidney through entry into epithelial cells of the distal nephron, inhibition of Glycogen Synthase Kinase-3β (GSK-3β) through phosphorylation...... on serine9 (pGSK-3β)and subsequent epithelial to mesenchymal dedifferentiation (EMT). GSK-3β immunoreactive protein was associated with collecting ducts in developing and adult human and rat kidney. Total GSK-3β protein abundance was stable in medulla while it decreased in cortex in the postnatal period...

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

Science.gov (United States)

2012-01-01

Background Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. Method We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability. Results In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W. Conclusion Hypercapnia with and without acidosis increased HPV during

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

Directory of Open Access Journals (Sweden)

Ketabchi Farzaneh

2012-01-01

Full Text Available Abstract Background Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV optimizes gas exchange during local acute (0-30 min, as well as sustained (> 30 min hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. Method We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate, and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min and endothelial permeability. Results In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA, a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS, decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc. This increase disappeared after administration of 1400 W. Conclusion Hypercapnia with and without acidosis

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction.

Science.gov (United States)

Ketabchi, Farzaneh; Ghofrani, Hossein A; Schermuly, Ralph T; Seeger, Werner; Grimminger, Friedrich; Egemnazarov, Bakytbek; Shid-Moosavi, S Mostafa; Dehghani, Gholam A; Weissmann, Norbert; Sommer, Natascha

2012-01-31

Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability. In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W. Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The

Cardiac-specific overexpression of catalase prevents diabetes-induced pathological changes by inhibiting NF-κB signaling activation in the heart.

Science.gov (United States)

Cong, Weitao; Ruan, Dandan; Xuan, Yuanhu; Niu, Chao; Tao, Youli; Wang, Yang; Zhan, Kungao; Cai, Lu; Jin, Litai; Tan, Yi

2015-12-01

Catalase is an antioxidant enzyme that specifically catabolizes hydrogen peroxide (H2O2). Overexpression of catalase via a heart-specific promoter (CAT-TG) was reported to reduce diabetes-induced accumulation of reactive oxygen species (ROS) and further prevent diabetes-induced pathological abnormalities, including cardiac structural derangement and left ventricular abnormity in mice. However, the mechanism by which catalase overexpression protects heart function remains unclear. This study found that activation of a ROS-dependent NF-κB signaling pathway was downregulated in hearts of diabetic mice overexpressing catalase. In addition, catalase overexpression inhibited the significant increase in nitration levels of key enzymes involved in energy metabolism, including α-oxoglutarate dehydrogenase E1 component (α-KGD) and ATP synthase α and β subunits (ATP-α and ATP-β). To assess the effects of the NF-κB pathway activation on heart function, Bay11-7082, an inhibitor of the NF-κB signaling pathway, was injected into diabetic mice, protecting mice against the development of cardiac damage and increased nitrative modifications of key enzymes involved in energy metabolism. In conclusion, these findings demonstrated that catalase protects mouse hearts against diabetic cardiomyopathy, partially by suppressing NF-κB-dependent inflammatory responses and associated protein nitration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Exogenous ethylene inhibits sprout growth in onion bulbs.

Science.gov (United States)

Bufler, Gebhard

2009-01-01

Exogenous ethylene has recently gained commercial interest as a sprouting inhibitor of onion bulbs. The role of ethylene in dormancy and sprouting of onions, however, is not known. A cultivar (Allium cepa 'Copra') with a true period of dormancy was used. Dormant and sprouting states of onion bulbs were treated with supposedly saturating doses of ethylene or with the ethylene-action inhibitor 1-methylcyclopropene (1-MCP). Initial sprouting was determined during storage at 18 degrees C by monitoring leaf blade elongation in a specific size class of leaf sheaths. Changes in ATP content and sucrose synthase activity in the sprout leaves, indicators of the sprouting state, were determined. CO(2) and ethylene production of onion bulbs during storage were recorded. Exogenous ethylene suppressed sprout growth of both dormant and already sprouting onion bulbs by inhibiting leaf blade elongation. In contrast to this growth-inhibiting effect, ethylene stimulated CO(2) production by the bulbs about 2-fold. The duration of dormancy was not significantly affected by exogenous ethylene. However, treatment of dormant bulbs with 1-MCP caused premature sprouting. Exogenous ethylene proved to be a powerful inhibitor of sprout growth in onion bulbs. The dormancy breaking effect of 1-MCP indicates a regulatory role of endogenous ethylene in onion bulb dormancy.

Inhibition of glycogen synthase kinase-3 reduces extension of the axonal leading process by destabilizing microtubules in cerebellar granule neurons.

Science.gov (United States)

Inami, Yoshihiro; Omura, Mitsuru; Kubota, Kenta; Konishi, Yoshiyuki

2018-07-01

Recent studies have uncovered various molecules that play key roles in neuronal morphogenesis. Nevertheless, the mechanisms underlying the neuron-type-dependent regulation of morphogenesis remain unknown. We have previously reported that inhibition of glycogen synthase kinase-3 (GSK3) markedly reduced axonal length of cerebellar granule neurons (CGNs) in a neuron-type-dependent manner. In the present study, we investigated the mechanisms by which the growth of CGN axons was severely suppressed upon GSK3 inhibition. Using time-lapse imaging of cultured CGNs at early morphogenesis, we found that extension of the leading process was severely inhibited by the pharmacological inhibition of GSK3. The rate of somal migration was also reduced with a GSK3 inhibitor in dissociated culture as well as in microexplant culture. In addition, CGNs ectopically expressed with a catalytically inactive mutant of GSK3 exhibited a migration defect in vivo. In axonal leading processes of CGNs, detyrosination and acetylation of α-tubulin, which are known to correlate with microtubule stability, were decreased by GSK3 inhibition. A photoconversion analysis found that inhibition of GSK3 increases the turnover of microtubules. Furthermore, in the presence of paclitaxel, a microtubule-stabilizing reagent, inhibition of GSK3 recovered the axonal leading process extension that was reduced by paclitaxel. Our results suggest that GSK3 supports the extension of axonal processes by stabilizing microtubules, contrary to its function in other neuron-types, lending mechanical insight into neuron-type-dependent morphological regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2013-12-01

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

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

Science.gov (United States)

Ostadhadi, Sattar; Akbarian, Reyhaneh; Norouzi-Javidan, Abbas; Nikoui, Vahid; Zolfaghari, Samira; Chamanara, Mohsen; Dehpour, Ahmad-Reza

2017-07-01

Gabapentin as an anticonvulsant drug also has beneficial effects in treatment of depression. Previously, we showed that acute administration of gabapentin produced an antidepressant-like effect in the mouse forced swimming test (FST) by a mechanism that involves the inhibition of nitric oxide (NO). Considering the involvement of NO in adenosine triphosphate (ATP)-sensitive potassium channels (K ATP ), in the present study we investigated the involvement of K ATP channels in antidepressant-like effect of gabapentin. Gabapentin at different doses (5-10 mg/kg) and fluoxetine (20 mg/kg) were administrated by intraperitoneal route, 60 and 30 min, respectively, before the test. To clarify the probable involvement of K ATP channels, mice were pretreated with K ATP channel inhibitor or opener. Gabapentin at dose 10 mg/kg significantly decreased the immobility behavior of mice similar to fluoxetine (20 mg/kg). Co-administration of subeffective dose (1 mg/kg) of glibenclamide (inhibitor of K ATP channels) with gabapentin (3 mg/kg) showed a synergistic antidepressant-like effect. Also, subeffective dose of cromakalim (opener of K ATP channels, 0.1 mg/kg) inhibited the antidepressant-like effect of gabapentin (10 mg/kg). None of the treatments had any impact on the locomotor movement. Our study, for the first time, revealed that antidepressant-like effect of gabapentin in mice is mediated by blocking the K ATP channels.

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

OpenAIRE

Steven Steven; Yoni F Syukriani; Julius B Dewanto

2016-01-01

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

Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; McGuire, James N

2004-01-01

The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s at 65 degrees C, respectively, for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes...... are hexamers. Vmax is determined as 440 micromol.min(-1).mg protein(-1) and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 microM, respectively, for the B. caldolyticus enzyme. The enzyme requires 50 mM Pi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2...

Effects of secretagogues on ATP levels and protein carboxyl methylation in rat brain synaptosomes

International Nuclear Information System (INIS)

Bjorndahl, J.M.; Rutledge, C.O.

1986-01-01

The influence of various substances which are known to alter free intracellular calcium concentrations on protein carboxyl methyltransferase (PCM) activity was investigated in rat brain synaptosomes. The synaptosomes were labeled with L-[ 3 H]methionine and the 3 H-methyl esters of proteins were formed from the methyl donor S-[ 3 H]adenosyl-L-methionine ([ 3 H]AdoMet). The calcium ionophore A23187 and ouabain decreased PCM activity and the decrease produced by A23187 was antagonized by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and MnCl 2 . On the other hand, ruthenium red, an inhibitor of calcium uptake, stimulated PCM activity. These data suggest that PCM activity is inversely related to the free cytoplasmic calcium concentration. Veratridine, A23187 and elevated potassium ions decreased the levels of ATP and [ 3 H]AdoMet. The A23187-mediated decrease in ATP levels and the reduced [ 3 H]AdoMet formation was antagonized by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and MnCl 2 . Inhibition of metabolic activity of the synaptosomes by NaCN led to: decreased ATP levels; inhibition of [3H]AdoMet formation; and inhibition of PCM activity. These data suggest that the decrease in protein methylation produced by secretagogues is associated with an increase in the concentration of free intracellular calcium which results in a decrease in the metabolically active pool of ATP. This leads to a decreased rate of AdoMet formation, a cosubstrate for PCM and a resultant decrease in PCM activity

Berberine improves glucose metabolism in diabetic rats by inhibition of hepatic gluconeogenesis.

Directory of Open Access Journals (Sweden)

Xuan Xia

2011-02-01

Full Text Available Berberine (BBR is a compound originally identified in a Chinese herbal medicine Huanglian (Coptis chinensis French. It improves glucose metabolism in type 2 diabetic patients. The mechanisms involve in activation of adenosine monophosphate activated protein kinase (AMPK and improvement of insulin sensitivity. However, it is not clear if BBR reduces blood glucose through other mechanism. In this study, we addressed this issue by examining liver response to BBR in diabetic rats, in which hyperglycemia was induced in Sprague-Dawley rats by high fat diet. We observed that BBR decreased fasting glucose significantly. Gluconeogenic genes, Phosphoenolpyruvate carboxykinase (PEPCK and Glucose-6-phosphatase (G6Pase, were decreased in liver by BBR. Hepatic steatosis was also reduced by BBR and expression of fatty acid synthase (FAS was inhibited in liver. Activities of transcription factors including Forkhead transcription factor O1 (FoxO1, sterol regulatory element-binding protein 1c (SREBP1 and carbohydrate responsive element-binding protein (ChREBP were decreased. Insulin signaling pathway was not altered in the liver. In cultured hepatocytes, BBR inhibited oxygen consumption and reduced intracellular adenosine triphosphate (ATP level. The data suggest that BBR improves fasting blood glucose by direct inhibition of gluconeogenesis in liver. This activity is not dependent on insulin action. The gluconeogenic inhibition is likely a result of mitochondria inhibition by BBR. The observation supports that BBR improves glucose metabolism through an insulin-independent pathway.

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

Science.gov (United States)

Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

2015-01-01

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline

NARCIS (Netherlands)

Diacon, Andreas H.; Pym, Alexander; Grobusch, Martin P.; de Los Rios, Jorge M.; Gotuzzo, Eduardo; Vasilyeva, Irina; Leimane, Vaira; Andries, Koen; Bakare, Nyasha; de Marez, Tine; Haxaire-Theeuwes, Myriam; Lounis, Nacer; Meyvisch, Paul; de Paepe, Els; van Heeswijk, Rolf P. G.; Dannemann, Brian; Rolla, Valeria; Dalcomo, Margreth; Gripp, Karla; Escada, Rodrigo; Tavares, Isabel; Borga, Liamar; Thomas, Aleyamma; Rekha, Banu; Nair, Dina; Chandrasekar, Chockalingam; Parthasarathy, Ramavaran Thiruvengadaraj; Sekhar, Gomathi; Ganesh, Krishnamoorthy; Rajagopalan, Krishnakumar; Rajapandian, Gangadevi; Dorairajalu, Rajendran; Sharma, Surendra Kumar; Banavaliker, Jayant; Kadhiravan, Tamilarasu; Gulati, Vinay; Mahmud, Hanif; Gupta, Arvind; Bhatnagar, Anuj; Jain, Vipin; Hari, Smriti; Gupta, Yogesh Kumar; Vaid, Ashok; Cirule, Andra; Dravniece, Gunta; Skripconoka, Vija; Kuksa, Liga; Kreigere, Edite; Ramos, Carlos Rafael Seas; Amat y Leon, Ivan Arapovic

2014-01-01

BACKGROUND Bedaquiline (Sirturo, TMC207), a diarylquinoline that inhibits mycobacterial ATP synthase, has been associated with accelerated sputum-culture conversion in patients with multidrug-resistant tuberculosis, when added to a preferred background regimen for 8 weeks. METHODS In this phase 2b

(-)-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer.

Science.gov (United States)

Crous-Masó, Joan; Palomeras, Sònia; Relat, Joana; Camó, Cristina; Martínez-Garza, Úrsula; Planas, Marta; Feliu, Lidia; Puig, Teresa

2018-05-11

(-)-Epigallocatechin 3-gallate (EGCG) is a natural polyphenol from green tea with reported anticancer activity and capacity to inhibit the lipogenic enzyme fatty acid synthase (FASN), which is overexpressed in several human carcinomas. To improve the pharmacological profile of EGCG, we previously developed a family of EGCG derivatives and the lead compounds G28, G37 and G56 were characterized in HER2-positive breast cancer cells overexpressing FASN. Here, diesters G28, G37 and G56 and two G28 derivatives, monoesters M1 and M2, were synthesized and assessed in vitro for their cytotoxic, FASN inhibition and apoptotic activities in MDA-MB-231 triple-negative breast cancer (TNBC) cells. All compounds displayed moderate to high cytotoxicity and significantly blocked FASN activity, monoesters M1 and M2 being more potent inhibitors than diesters. Interestingly, G28, M1, and M2 also diminished FASN protein expression levels, but only monoesters M1 and M2 induced apoptosis. Our results indicate that FASN inhibition by such polyphenolic compounds could be a new strategy in TNBC treatment, and highlight the potential anticancer activities of monoesters. Thus, G28, G37, G56, and most importantly M1 and M2, are anticancer candidates (alone or in combination) to be further characterized in vitro and in vivo.

ATP signals

DEFF Research Database (Denmark)

Novak, Ivana

2016-01-01

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

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts.

Science.gov (United States)

Pizer, E S; Thupari, J; Han, W F; Pinn, M L; Chrest, F J; Frehywot, G L; Townsend, C A; Kuhajda, F P

2000-01-15

A biologically aggressive subset of human breast cancers and other malignancies is characterized by elevated fatty-acid synthase (FAS) enzyme expression, elevated fatty acid (FA) synthesis, and selective sensitivity to pharmacological inhibition of FAS activity by cerulenin or the novel compound C75. In this study, inhibition of FA synthesis at the physiologically regulated step of carboxylation of acetyl-CoA to malonyl-CoA by 5-(tetradecyloxy)-2-furoic acid (TOFA) was not cytotoxic to breast cancer cells in clonogenic assays. FAS inhibitors induced a rapid increase in intracellular malonyl-CoA to several fold above control levels, whereas TOFA reduced intracellular malonyl-CoA by 60%. Simultaneous exposure of breast cancer cells to TOFA and an FAS inhibitor resulted in significantly reduced cytotoxicity and apoptosis. Subcutaneous xenografts of MCF7 breast cancer cells in nude mice treated with C75 showed FA synthesis inhibition, apoptosis, and inhibition of tumor growth to less than 1/8 of control volumes, without comparable toxicity in normal tissues. The data suggest that differences in intermediary metabolism render tumor cells susceptible to toxic fluxes in malonyl-CoA, both in vitro and in vivo.

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

Science.gov (United States)

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

2011-01-01

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

A trypsin inhibitor from Tecoma stans leaves inhibits growth and promotes ATP depletion and lipid peroxidation in Candida albicans and Candida krusei

Directory of Open Access Journals (Sweden)

Leydianne Leite de Siqueira Patriota

2016-04-01

Full Text Available Tecoma stans (yellow elder has shown medicinal properties and antimicrobial activity. Previous reports on antifungal activity of T. stans preparations and presence of trypsin inhibitor activity from T. stans leaves stimulated the investigation reported here. In this work, we proceeded to the purification and characterization of a trypsin inhibitor (TesTI, which was investigated for anti-Candida activity. Finally, in order to determine the potential of TesTI as a new natural chemotherapeutic product, its cytotoxicity to human peripheral blood mononuclear cells (PBMCs was evaluated. TesTI was isolated from saline extract by ammonium sulphate fractionation followed by ion exchange and gel filtration chromatographies. Antifungal activity was evaluated by determining the minimal inhibitory (MIC and fungicide (MFC concentrations using fungal cultures containing only yeast form or both yeast and hyphal forms. Candida cells treated with TesTI were evaluated for intracellular ATP levels and lipid peroxidation. Cytotoxicity of TesTI to PBMCs was evaluated by MTT assay. TesTI (39.8 kDa, pI 3.41, Ki 43 nM inhibited similarly the growth of both C. albicans and C. krusei culture types at MIC of 100 µg/mL. The MFCs were 200 µg/mL for C. albicans and C. krusei. Time-response curves revealed that TesTI (at MIC was more effective at inhibiting the replication of C. albicans cells. At MIC, TesTI promoted reduction of ATP levels and lipid peroxidation in the Candida cells, being not cytotoxic to PBMCs. In conclusion, TesTI is an antifungal agent against C. albicans and C. krusei, without toxicity to human cells.

Chronic inhibition of glycogen synthase kinase-3 protects against rotenone-induced cell death in human neuron-like cells by increasing BDNF secretion.

Science.gov (United States)

Giménez-Cassina, Alfredo; Lim, Filip; Díaz-Nido, Javier

2012-12-07

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. Likewise, activation of glycogen synthase kinase-3 (GSK-3) has been proposed to play an important role in neurodegeneration. This multifunctional protein kinase is involved in a number of cellular functions and we previously showed that chronic inhibition of GSK-3 protects neuronal cells against mitochondrial dysfunction-elicited cell death, through a mechanism involving increased glucose metabolism and the translocation of hexokinase II (HKII) to mitochondria. Here, we sought to gain deeper insight into the molecular basis of this neuroprotection. We found that chronic inhibition of GSK-3, either genetically or pharmacologically, elicited a marked increase in brain-derived neurotrophic factor (BDNF) secretion, which in turn conferred resistance to mitochondrial dysfunction through subcellular re-distribution of HKII. These results define a molecular pathway through which chronic inhibition of GSK-3 may protect neuronal cells from death. Moreover, they highlight the potential benefits of enhanced neurotrophic factor secretion as a therapeutic approach to treat neurodegenerative diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

Froehlich, D.

1978-01-01

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

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

Science.gov (United States)

Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-01-01

Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

Science.gov (United States)

Chen, Ying-Bei; Aon, Miguel A; Hsu, Yi-Te; Soane, Lucian; Teng, Xinchen; McCaffery, J Michael; Cheng, Wen-Chih; Qi, Bing; Li, Hongmei; Alavian, Kambiz N; Dayhoff-Brannigan, Margaret; Zou, Shifa; Pineda, Fernando J; O'Rourke, Brian; Ko, Young H; Pedersen, Peter L; Kaczmarek, Leonard K; Jonas, Elizabeth A; Hardwick, J Marie

2011-10-17

Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis.

Science.gov (United States)

Gas-Pascual, Elisabet; Simonovik, Biljana; Heintz, Dimitri; Bergdoll, Marc; Schaller, Hubert; Bach, Thomas J

2015-08-01

The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC-MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

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

Science.gov (United States)

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

2000-01-18

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

Glycogen synthase kinase-3 inhibition sensitizes human induced pluripotent stem cells to thiol-containing antioxidants induced apoptosis.

Science.gov (United States)

Tu, Chengyi; Xu, Robert; Koleti, Meghana; Zoldan, Janet

2017-08-01

Inhibition of glycogen synthase kinase 3 (GSK3) is an extensively used strategy to activate Wnt pathway for pluripotent stem cell (PSC) differentiation. However, the effects of such inhibition on PSCs, besides upregulating the Wnt pathway, have rarely been investigated despite that GSK3 is broadly involved in other cellular activities such as insulin signaling and cell growth/survival regulation. Here we describe a previously unknown synergistic effect between GSK3 inhibition (e.g., Chir99021 and LY2090314) and various normally non-toxic thiol-containing antioxidants (e.g., N-acetylcysteine, NAC) on the induction of apoptosis in human induced pluripotent stem cells (iPSCs). Neither Chir99021 nor the antioxidants individually induced significant apoptosis, whereas their combined treatment resulted in rapid and extensive apoptosis, with substantial caspase 3 activity observed within 3h and over 90% decrease in cell viability after 24h. We confirmed the generality of this phenomenon with multiple independent iPSCs lines, various thiol-based antioxidants and distinct GSK3 inhibitors. Mechanistically, we demonstrated that rapamycin treatment could substantially reduce cell death, suggesting the critical role of mammalian target of rapamycin (mTOR). Akt dysregulation was also found to partially contribute to cell apoptosis but was not the primary cause. Further, this coordinated proapoptotic effect was not detected in mouse ESCs but was present in another human cells line: a breast cancer cell line (MDA-MB-231). Given the wide use of GSK3 inhibition in biomedical research: from iPSC differentiation to cancer intervention and the treatment of neuronal diseases, researchers can potentially take advantage of or avoid this synergistic effect for improved experimental or clinical outcome. Copyright © 2017. Published by Elsevier B.V.

Influence of age-related changes in nitric oxide synthase-expressing neurons in the rat supraoptic nucleus on inhibition of salivary secretion.

Science.gov (United States)

Tanaka, Takehiko; Tamada, Yoshitaka; Suwa, Fumihiko

2008-02-01

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Hydrogen sulfide-mediated regulation of contractility in the mouse ileum with electrical stimulation: roles of L-cysteine, cystathionine β-synthase, and K+ channels.

Science.gov (United States)

Yamane, Satoshi; Kanno, Toshio; Nakamura, Hiroyuki; Fujino, Hiromichi; Murayama, Toshihiko

2014-10-05

Hydrogen sulfide (H2S) is considered to be a signaling molecule. The precise mechanisms underlying H2S-related events, including the producing enzymes and target molecules in gastrointestinal tissues, have not been elucidated in detail. We herein examined the involvement of H2S in contractions induced by repeated electrical stimulations (ES). ES-induced contractions were neurotoxin-sensitive and increased by aminooxyacetic acid, an inhibitor of cystathionine β-synthase (CBS) and cystathionine γ-lyase, but not by D,L-propargylglycine, a selective inhibitor of cystathionine γ-lyase, in an ES trial-dependent manner. ES-induced contractions were markedly decreased in the presence of L-cysteine. This response was inhibited by aminooxyacetic acid and an antioxidant, and accelerated by L-methionine, an activator of CBS. The existence of CBS was confirmed. NaHS transiently inhibited ES- and acetylcholine-induced contractions, and sustainably decreased basal tone for at least 20 min after its addition. The treatment with glibenclamide, an ATP-sensitive K+ channel blocker, reduced both the L-cysteine response and NaHS-induced inhibition of contractions. The NaHS-induced decrease in basal tone was inhibited by apamin, a small conductance Ca2+-activated K+ channel blocker. These results suggest that H2S may be endogenously produced via CBS in ES-activated enteric neurons, and regulates contractility via multiple K+ channels in the ileum. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibition of glycogen synthase kinase-3β counteracts ligand-independent activity of the androgen receptor in castration resistant prostate cancer.

Directory of Open Access Journals (Sweden)

Stefanie V Schütz

Full Text Available In order to generate genomic signals, the androgen receptor (AR has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.

Glossogyne Tenuifolia Enhances Posttranslational S-Nitrosylation of Proteins in Vascular Endothelial Cells

Directory of Open Access Journals (Sweden)

Chao-Ping Wang

2011-06-01

Full Text Available Glossogyne tenuifolia (GT is a traditional Chinese herb that possesses strong antioxidant activity and protects against endothelial cell (EC injury by inhibition of free reactive oxygen species (ROS. The aim of this study was to elucidate the mechanisms by which GT prevents endothelial injury using a proteomics approach. We used a sensitive method to analyze the S- nitrosoproteins utilizing a modified biotin-switch method in order to detect the possible effects of GT on protein posttranslational modification. After treatment of vascular ECs with GT, two proteins HspA9 (IS1, beta-actin (IS2 were observed to have increased posttranslational S-nitrosylation, whereas seven proteins, vimentin (DS2, DS3 and DS5, tropomyosin 3, 4 (DS6 and DS7 and oxidative phosphorylation protein such as ATP synthase, F1 complex (DS1 and 80K-H protein (DS4, were found to have decreased posttranslational S-nitrosylation. Due to S-nitrosylation of HspA9 causing the reduction of intracellular ROS and S-nitrosylation of ATP synthase interfering with ATP production and ROS formation, our study may indicate a novel mechanism in which GT protects EC injury by the inhibition of oxidative reaction.

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.

Effect of phosphodiesterase inhibitors on human arteries in vitro

NARCIS (Netherlands)

Vroom, M. B.; Pfaffendorf, M.; van Wezel, H. B.; van Zwieten, P. A.

1996-01-01

In the present study, we investigated if the relaxant effects of phosphodiesterase (PDE) III inhibitors on human vessels could be inhibited by a nitric oxide synthase blocker, L-NAME, or by a blocker of ATP-sensitive potassium channels (KATP), glibenclamide. The experiments were performed using an

(−-Epigallocatechin 3-Gallate Synthetic Analogues Inhibit Fatty Acid Synthase and Show Anticancer Activity in Triple Negative Breast Cancer

Directory of Open Access Journals (Sweden)

Joan Crous-Masó

2018-05-01

Full Text Available (−-Epigallocatechin 3-gallate (EGCG is a natural polyphenol from green tea with reported anticancer activity and capacity to inhibit the lipogenic enzyme fatty acid synthase (FASN, which is overexpressed in several human carcinomas. To improve the pharmacological profile of EGCG, we previously developed a family of EGCG derivatives and the lead compounds G28, G37 and G56 were characterized in HER2-positive breast cancer cells overexpressing FASN. Here, diesters G28, G37 and G56 and two G28 derivatives, monoesters M1 and M2, were synthesized and assessed in vitro for their cytotoxic, FASN inhibition and apoptotic activities in MDA-MB-231 triple-negative breast cancer (TNBC cells. All compounds displayed moderate to high cytotoxicity and significantly blocked FASN activity, monoesters M1 and M2 being more potent inhibitors than diesters. Interestingly, G28, M1, and M2 also diminished FASN protein expression levels, but only monoesters M1 and M2 induced apoptosis. Our results indicate that FASN inhibition by such polyphenolic compounds could be a new strategy in TNBC treatment, and highlight the potential anticancer activities of monoesters. Thus, G28, G37, G56, and most importantly M1 and M2, are anticancer candidates (alone or in combination to be further characterized in vitro and in vivo.

4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

International Nuclear Information System (INIS)

Kultti, Anne; Pasonen-Seppaenen, Sanna; Jauhiainen, Marjo; Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I.

2009-01-01

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

Energy Technology Data Exchange (ETDEWEB)

Kultti, Anne, E-mail: anne.kultti@uku.fi [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Pasonen-Seppaenen, Sanna [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Jauhiainen, Marjo [Department of Pharmaceutical Chemistry, University of Kuopio, FIN-70211 Kuopio (Finland); Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I. [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland)

2009-07-01

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

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

DEFF Research Database (Denmark)

Kowal, Justyna Magdalena

release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological......ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......, particularly during Ca2+ stress conditions. In conclusion, these studies demonstrate a complex regulation of purinergic signalling in exocrine pancreas. A crucial role for duct cells in mediating extracellular nucleotides homeostasis, involving ATP release, subsequent hydrolysis and conversion via...

Is the ATPase from halobacterium saccharovorum an evolutionary relic?

Science.gov (United States)

Hochstein, L. I.; Altekar, W.; Kristjansson, H.

1986-01-01

The ATP Synthase Complex present in the membranes of mitochondria, chloroplasts or bacteria is composed of 2 sectors: FO, an integral membrane protein consisting of 3 subunits mediating proton translocation across the membrane and F1, the catalytic component composed of 5 non-identical subunits. The apparent early origin of the ATP Synthase Complex, as implied by its ubiquitous distribution, seems inconsistent with its structural and functional complexity and raises the question if simpler versions of the ATP Synthase exist. Such an ATP Synthase has been searched for in various Archaebacteria. A purified halobacterial ATPase activity which possesses certain properties consistent with those of an ATP Synthase but which has a different subunit structure is described.

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.

Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

Science.gov (United States)

Xu, Yang; Wang, Guan; Li, Chunjie; Zhang, Min; Zhao, Hang; Sheng, Jun; Shi, Wei

2012-01-01

Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4. PMID:22837686

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

Science.gov (United States)

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

2012-08-01

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

Requirement of phosphorylatable endothelial nitric oxide synthase at Ser-1177 for vasoinhibin-mediated inhibition of endothelial cell migration and proliferation in vitro.

Science.gov (United States)

García, Celina; Nuñez-Anita, Rosa Elvira; Thebault, Stéphanie; Arredondo Zamarripa, David; Jeziorsky, Michael C; Martínez de la Escalera, Gonzalo; Clapp, Carmen

2014-03-01

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16 K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylation of eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.

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

International Nuclear Information System (INIS)

Penefsky, H.S.

1985-01-01

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

Factors influencing gene silencing of granule-bound starch synthase in potato

NARCIS (Netherlands)

Heilersig, H.J.B.

2005-01-01

In the past, antisense RNA technology was used to modify the composition of potato tuber starch. Potato starch comprises amylose and amylopectin, polymers of glucose. Amylose production in potato is completely dependent on the presence of granule-bound starch synthase I (GBSSI). Inhibition of GBSSI

Tyrosinase inhibition due to interaction of homocyst(e)ine with copper: the mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency.

Science.gov (United States)

Reish, O; Townsend, D; Berry, S A; Tsai, M Y; King, R A

1995-01-01

Deficiency of cystathionine beta-synthase (CBS) is a genetic disorder of transsulfuration resulting in elevated plasma homocyst(e)ine and methionine and decreased cysteine. Affected patients have multisystem involvement, which may include light skin and hair. Reversible hypopigmentation in treated homocystinuric patients has been infrequently reported, and the mechanism is undefined. Two CBS-deficient homocystinuric patients manifested darkening of their hypopigmented hair following treatment that decreased plasma homocyst(e)ine. We hypothesized that homocyst(e)ine inhibits tyrosinase, the major pigment enzyme. The activity of tyrosinase extracted from pigmented human melanoma cells (MNT-1) that were grown in the presence of homocysteine was reduced in comparison to that extracted from cells grown without homocysteine. Copper sulfate restored homocyst(e)ine-inhibited tyrosinase activity when added to the culture cell media at a proportion of 1.25 mol of copper sulfate per 1 mol of DL-homocysteine. Holo-tyrosinase activity was inhibited by adding DL-homocysteine to the assay reaction mixture, and the addition of copper sulfate to the reaction mixture prevented this inhibition. Other tested compounds, L-cystine and betaine did not affect tyrosinase activity. Our data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocyst(e)ine and that the probable mechanism of this inhibition is the interaction of homocyst(e)ine with copper at the active site of tyrosinase. Images Figure 1 PMID:7611281

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

Energy Technology Data Exchange (ETDEWEB)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-08-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

International Nuclear Information System (INIS)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-01-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

International Nuclear Information System (INIS)

Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

1988-01-01

Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca 2+ -dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca 2+ -independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10 -6 M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10 -4 M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 μM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [γ- 32 P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase

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

KAUST Repository

Jourdain, P.

2016-02-19

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

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

KAUST Repository

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

2016-01-01

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

Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

OpenAIRE

Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

2017-01-01

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

Science.gov (United States)

Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

2007-01-01

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures

Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition.

Science.gov (United States)

Jones, Sara E; Whitehead, Kristi; Saulnier, Delphine; Thomas, Carissa M; Versalovic, James; Britton, Robert A

2011-01-01

Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required or human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuter immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

Science.gov (United States)

Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2015-07-01

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

International Nuclear Information System (INIS)

Du, Z.; Boyer, P.D.

1990-01-01

Washed chloroplast thylakoid membranes upon exposure to [ 3 H]ADP retain in tightly bound [ 3 H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg 2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [ 3 H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg 2+ - and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22 degree C and of about 15 s at 37 degree C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg 2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [ 3 H]ADP parallels the onset of ATPase activity, although some [ 3 H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [ 3 H]ADP being at a catalytic site and being replaced as this Mg 2+ - and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P i binding site of the enzyme-ADP-Mg 2+ complex to give a form more readily activated by ATP binding at an alternative site

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

Science.gov (United States)

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

2016-07-01

In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of the Vacuolar-like ATPase from Halobacterium saccharovorum by Thiol Reagents: Evidence for Different Functional Thiols

Science.gov (United States)

Hochstein, L. I.; Stanlotter, H.; Emrich, E.; Morrison, David (Technical Monitor)

1994-01-01

N-Ethylmaleimide (NEM) inhibited the vacuolar-like ATPase from Halobacterium saccharovorum (K(sub i) approximately 1 mM) by modifying one or more of the thiols located on the largest of the subunit. ATP protected against inhibition and coincidentally prevented NEM binding which suggested that NEM acts at or near the catalytic site. p-Chloromercuriphenylsulfonate (PCMS) also inhibited this ATPase (K(sub i) approximately 90 microM). ATP did not protect against PCMS inhibition. Dithiothreitol (DTT) partially reversed PCMS inhibition and restored approximately half of the initial activity of 90% inhibited enzyme. DTT did not restore activity of the NEM-inhibited enzyme or the PCMS-inhibited enzyme when it was subsequently incubated with NEM. The failure of ATP to protect against PCMS inhibition and the inability of DTT to restore activity of enzyme incubated in the presence of PCMS and NEM suggests these reagents react with different thiols and that the PCMS-sensitive thiol may have a structural role.

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

International Nuclear Information System (INIS)

Kun, Ernest; Kirsten, Eva; Hakam, Alaeddin; Bauer, Pal I.; Mendeleyev, Jerome

2008-01-01

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

Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse Inhibition of brain citrate synthase activity in an animal model of sepsis

Directory of Open Access Journals (Sweden)

Giselli Scaini

2011-06-01

fisiopatologia desta doença.OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS. Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.

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

Science.gov (United States)

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

2009-01-01

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

Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

Directory of Open Access Journals (Sweden)

Shailendra P. Singh

2015-08-01

Full Text Available Glycogen synthase kinase-3β (GSK3β is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

Ballast water compliance monitoring: A new application for ATP

Science.gov (United States)

Lo Curto, A.; Stehouwer, P.; Gianoli, C.; Schneider, G.; Raymond, M.; Bonamin, V.

2018-03-01

The coming into force of the USCG ballast water regulations and the IMO ballast water management convention resulted in the development of several technologies approved for the treatment of ballast water. To ensure compliance of these technologies, the development of rapid and robust analysis methods was necessary. In collaboration with the SGS Group (Switzerland) and LuminUltra (Canada), Aqua-tools (France) has developed an innovative Ballast Water Treatment Monitoring (BWTM) kit for rapid onboard testing. The affordable kit provides results in less than 1 h, is easy to use and durable ensuring that the ballast water treatment system on the ship is fully compliant with the discharge standards upon arrival in port. The core of this method is a combination of high-quality reagents (lysis solution and ATP 2G Luminase™ enzyme) not inhibited by salinity and a patented fast homogenizing method for ATP extraction developed for a higher ATP recovery from zooplankton and phytoplankton. Compared to traditional analysis methods, the BWTM Kit provides fast and accurate results for all three fractions of microorganisms (≥ 50 μm, ≥ 10 ÷ < 50 μm and bacteria). Preliminary tests carried out in cooperation with SGS showed that the proposed method was able to detect onboard the efficiency of the treatment systems used. Compliance limits were established for all size fractions and a correlation between the standard methods (microscopy, plate count, MPN) and ATP was evaluated. The BWTM kit can provide a fast indication of compliance or gross exceedance. The rare borderline cases, when encountered, of course require additional confirmation.

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Kelley, M.J.; Carman, G.M.

1987-01-01

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase was purified 2300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism

Functional reconstitution of an ATP-driven Ca2+-transport system from the plasma membrane of Commelina communis L

International Nuclear Information System (INIS)

Graef, P.; Weiler, E.W.

1990-01-01

The protein(s) that constitute(s) the ATP-driven Ca 2+ -translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca 2+ -transport system was studied using ATP-driven 45 Ca 2+ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid and ATP improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K m for Ca 2+ inhibition by relatively high levels of vanadate and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B and had a low apparent K m for ATP levels of the Ca 2+ -ionophore A 23187 instantaneously discharged 90% of the Ca 2+ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca 2+ -transport in the reconstituted system was thus primary active, through a Ca 2+ -translocating ATPase

Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show Anti-HCV Activity and in Vivo Target Modulation.

Science.gov (United States)

Oslob, Johan D; Johnson, Russell J; Cai, Haiying; Feng, Shirley Q; Hu, Lily; Kosaka, Yuko; Lai, Julie; Sivaraja, Mohanram; Tep, Samnang; Yang, Hanbiao; Zaharia, Cristiana A; Evanchik, Marc J; McDowell, Robert S

2013-01-10

Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo.

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

Science.gov (United States)

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

1986-02-15

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

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

Science.gov (United States)

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

2016-12-01

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

ATP-induced changes in rat skeletal muscle contractility.

Science.gov (United States)

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

2015-01-01

the European Convention for the Protection of Vertebrate Animals used in scientific experiments. All agents used in the study were supplied by Sigma Chemical Company Ltd. (UK), Tocris Cookson and Research Biochemicals International (USA). The concentration of 100 μM for adenosine is close to saturation [1], and for its predecessor ATP this concentration is created after the passage of a pulse through the synapse [2]. We used this concentration of purines to study the mechanism of action of adenosine and ATP on neuromuscular synapse.The effect of adenosine was partially inhibited in the presence of 100 μM 8-SPT, an antagonist of adenosine receptors. The contraction force of "fast" and "slow" rat skeletal muscles was raised by half in the presence of norepinephrine. In the presence of norepinephrine adenosine exerted its effect fully, but ATP by half reduced its depressor effect on the contraction force of both muscles. 1. Norepinephrine increases half times of the reduction of "fast" and "slow" skeletal muscle.2. In the presence of norepinephrine, inhibitory effect of adenosine on contraction force is maintained.3. Inhibitory effect of ATP on contraction force of studied skeletal muscles becomes twice less pronounced in the presence of norepinephrine.We think that reduction of ATP depressive effect on the skeletal muscle by norepinephrine may be an adaptive response to acute stress.

Longevity in vivo of primary cell wall cellulose synthases.

Science.gov (United States)

Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

2018-02-01

Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats

DEFF Research Database (Denmark)

Graebe, M.; Brond, L.; Christensen, S.

2004-01-01

The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation...... (CBL). Three weeks of daily sodium balance studies showed that CBL rats developed sodium retention compared with sham-operated rats and that l-NAME treatment dose dependently deteriorated cumulative sodium balance by reducing urinary sodium excretion. Five weeks after CBL, renal clearance studies were...

Monoterpene synthases from common sage (Salvia officinalis)

Energy Technology Data Exchange (ETDEWEB)

Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

1999-01-01

cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

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

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

Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

International Nuclear Information System (INIS)

Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin; Fu, Xinlu; Shen, Feihai; Huang, Zhiying

2016-01-01

Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

Energy Technology Data Exchange (ETDEWEB)

Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Fu, Xinlu [Laboratory Animals Center, Sun Yat-sen University, Guangzhou 510006 (China); Shen, Feihai, E-mail: shenfh3@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Huang, Zhiying, E-mail: hzhiying@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China)

2016-12-15

Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

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

International Nuclear Information System (INIS)

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

1986-01-01

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

β-chain of ATP synthase as a lipophorin binding protein and its role in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae).

Science.gov (United States)

Fruttero, Leonardo L; Demartini, Diogo R; Rubiolo, Edilberto R; Carlini, Célia R; Canavoso, Lilián E

2014-09-01

Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The β subunit of the ATP synthase complex (β-ATPase) was identified as a lipophorin binding protein. β-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that β-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and β-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of β-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the β-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-β-ATPase association in the transfer of lipids. In addition, the β-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

Science.gov (United States)

Somerville, Chris R [Portola Valley, CA; Scheible, Wolf [Golm, DE

2007-07-10

Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

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

DEFF Research Database (Denmark)

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

2015-01-01

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

Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase.

Science.gov (United States)

Cingolani, Francesca; Simbari, Fabio; Abad, Jose Luis; Casasampere, Mireia; Fabrias, Gemma; Futerman, Anthony H; Casas, Josefina

2017-08-01

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

Directory of Open Access Journals (Sweden)

Mamaghani Shadi

2009-04-01

Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard

Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

International Nuclear Information System (INIS)

Mamaghani, Shadi; Patel, Satish; Hedley, David W

2009-01-01

Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-X L , and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard chemotherapy agent gemcitabine. This lack of synergy might be

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...

Protection against Mitochondrial and Metal Toxicity Depends on Functional Lipid Binding Sites in ATP13A2

Directory of Open Access Journals (Sweden)

Shaun Martin

2016-01-01

Full Text Available The late endo-/lysosomal P-type ATPase ATP13A2 (PARK9 is implicated in Parkinson’s disease (PD and Kufor-Rakeb syndrome, early-onset atypical Parkinsonism. ATP13A2 interacts at the N-terminus with the signaling lipids phosphatidic acid (PA and phosphatidylinositol (3,5 bisphosphate (PI(3,5P2, which modulate ATP13A2 activity under cellular stress conditions. Here, we analyzed stable human SHSY5Y cell lines overexpressing wild-type (WT or ATP13A2 mutants in which three N-terminal lipid binding sites (LBS1–3 were mutated. We explored the regulatory role of LBS1–3 in the cellular protection by ATP13A2 against mitochondrial stress induced by rotenone and found that the LBS2-3 mutants displayed an abrogated protective effect. Moreover, in contrast to WT, the LBS2 and LBS3 mutants responded poorly to pharmacological inhibition of, respectively, PI(3,5P2 and PA formation. We further demonstrate that PA and PI(3,5P2 are also required for the ATP13A2-mediated protection against the toxic metals Mn2+, Zn2+, and Fe3+, suggesting a general lipid-dependent activation mechanism of ATP13A2 in various PD-related stress conditions. Our results indicate that the ATP13A2-mediated protection requires binding of PI(3,5P2 to LBS2 and PA to LBS3. Thus, targeting the N-terminal lipid binding sites of ATP13A2 might offer a therapeutic approach to reduce cellular toxicity of various PD insults including mitochondrial stress.

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.

Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis

NARCIS (Netherlands)

Heeringa, P; van Goor, H; Itoh-Lindstrom, Y; Maeda, N; Falk, RJ; Assmann, KJM; Kallenberg, CGM; Jennette, JC

Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study

EST Table: FS737333 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available FS737333 E_FL_bmmt_08I09_R_0 10/09/28 68 %/182 aa ref|NP_059479.1| ATP synthase F0 ...subunit 6 [Bombyx mori] gb|AAF73768.1|AF149768_12 ATPase 6 [Bombyx mori] gb|AAK85722.1| ATPase subunit 6 [Bo....1| ATP synthase F0 subunit 6 [Bombyx mori] gb|ADE18460.1| ATP synthase F0 subunit 6 [Bombyx mori] gb|ADE18473

Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

DEFF Research Database (Denmark)

Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva

2005-01-01

The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and...

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

Science.gov (United States)

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

2011-07-01

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

Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing upregulation of inducible nitric oxide synthase.

Science.gov (United States)

Zucker, Stephen D; Vogel, Megan E; Kindel, Tammy L; Smith, Darcey L H; Idelman, Gila; Avissar, Uri; Kakarlapudi, Ganesh; Masnovi, Michelle E

2015-11-15

Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression. Copyright © 2015 the American Physiological Society.

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Cooperation for Better Inhibiting.

Science.gov (United States)

Novoa, Eva Maria; Ribas de Pouplana, Lluís

2015-06-18

Cladosporin is an antimalarial drug that acts as an ATP-mimetic to selectively inhibit Plasmodium lysyl-tRNA synthetase. Using multiple crystal structures, Fang et al. (2015) reveal in this issue of Chemistry & Biology the fascinating mechanism responsible for cladosporin selectivity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria - An amazing defence tool against hyperosmotic stress

Directory of Open Access Journals (Sweden)

Daniela eTrono

2015-12-01

Full Text Available In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about fifteen years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP. Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous and tissues/organs (etiolated and green have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane towards the matrix, so collapsing membrane potential (ΔΨ, the main component of the protonmotive force (Δp in plant mitochondria; moreover, cooperation between PmitoKATP and the K+/H+ antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate

The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia.

Science.gov (United States)

Sgarbi, G; Barbato, S; Costanzini, A; Solaini, G; Baracca, A

2018-02-01

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF 1 , that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O 2 ) unexpectedly showed that Δψ m is preserved independently of the presence of IF 1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing Δμ Η + with the FCCP uncoupler, the IF 1 -silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF 1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF 1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2018-06-02

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

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

DEFF Research Database (Denmark)

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

2012-01-01

The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B......, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type...

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

Directory of Open Access Journals (Sweden)

Ip Virginia

2010-09-01

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

The ATP required for potentiation of skeletal muscle contraction is released via pannexin hemichannels.

Science.gov (United States)

Riquelme, Manuel A; Cea, Luis A; Vega, José L; Boric, Mauricio P; Monyer, Hannah; Bennett, Michael V L; Frank, Marina; Willecke, Klaus; Sáez, Juan C

2013-12-01

During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Effect of selective inhibition of renal inducible nitric oxide synthase on renal blood flow and function in experimental hyperdynamic sepsis.

Science.gov (United States)

Ishikawa, Ken; Calzavacca, Paolo; Bellomo, Rinaldo; Bailey, Michael; May, Clive N

2012-08-01

Nitric oxide plays an important role in the control of renal blood flow and renal function. In sepsis, increased levels of inducible nitric oxide synthase produce excessive nitric oxide, which may contribute to the development of acute kidney injury. We, therefore, examined the effects of intrarenal infusion of selective inducible nitric oxide synthase inhibitors in a large animal model of hyperdynamic sepsis in which acute kidney injury occurs in the presence of increased renal blood flow. Prospective crossover randomized controlled interventional studies. University-affiliated research institute. Twelve unilaterally nephrectomized Merino ewes. Infusion of a selective (1400W) and a partially selective inducible nitric oxide synthase inhibitor (aminoguanidine) into the renal artery for 2 hrs after the induction of sepsis, and comparison with a nonselective inhibitor (Nω-nitro-L-arginine methyl ester). In sheep with nonhypotensive hyperdynamic sepsis, creatinine clearance halved (32 to 16 mL/min, ratio [95% confidence interval] 0.51 [0.28-0.92]) despite increased renal blood flow (241 to 343 mL/min, difference [95% confidence interval] 102 [78-126]). Infusion of 1400W did not change renal blood flow, urine output, or creatinine clearance, whereas infusion of Nω-nitro-L-arginine methyl ester and a high dose of aminoguanidine normalized renal blood flow, but did not alter creatinine clearance. In hyperdynamic sepsis, intrarenal infusion of a highly selective inducible nitric oxide synthase inhibitor did not reduce the elevated renal blood flow or improve renal function. In contrast, renal blood flow was reduced by infusion of a nonselective NOS inhibitor or a high dose of a partially selective inducible nitric oxide synthase inhibitor. The renal vasodilatation in septic acute kidney injury may be due to nitric oxide derived from the endothelial and neural isoforms of nitric oxide synthase, but their blockade did not restore renal function.

Effects of inhibitors of protein kinase C and NO-synthase on the radiation-induced cytogenetic adaptive response in Chinese hamster cells in culture

International Nuclear Information System (INIS)

Gil'yano, N.Ya.; Bondarev, G.N.; Bikineeva, E.G.; Krasotskaya, G.I.; Noskin, L.A.

2001-01-01

The effect of the serine-threonin kinase inhibitor - staurosporine and inhibitor of NO-synthase - L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. It is shown that staurosporine and L-NAME inhibit cytogenetic adaptive response induced by β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-rays-induced chromosome aberration yield also. Study confirms the role of protein kinase C in induction of the adaptive response and participation of NO-synthase in this process is noticed for the first time [ru

On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

Energy Technology Data Exchange (ETDEWEB)

Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

1990-01-16

Washed chloroplast thylakoid membranes upon exposure to ({sup 3}H)ADP retain in tightly bound ({sup 3}H)ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg{sup 2+} results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound ({sup 3}H)ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg{sup 2+}- and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22{degree}C and of about 15 s at 37{degree}C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg{sup 2+} and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound ({sup 3}H)ADP parallels the onset of ATPase activity, although some ({sup 3}H)ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound ({sup 3}H)ADP being at a catalytic site and being replaced as this Mg{sup 2+}- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P{sub i} binding site of the enzyme-ADP-Mg{sup 2+} complex to give a form more readily activated by ATP binding at an alternative site.

Fluorescence and chemiluminescence behavior of distyrylbenzene bearing two arms of dipicolylaminomethyl groups: Interactions with zinc ion and ATP

Science.gov (United States)

Motoyoshiya, Jiro; Wada, Jun-ya; Itoh, Keiko; Wakabayashi, Kazuaki; Maruyama, Takayuki; Ono, Kazuki; Fukasawa, Kota; Fujimoto, Tetsuya; Akaiwa, Yuji; Nonaka, Eiji

2018-04-01

The absorption and fluorescence spectral study of the distyrylbenzene bearing two arms of the dipicolylaminomethyl groups, the effective ligands for Zn2+, was studied in the presence of Zn2+ and ATP. Upon complexation of the distyrylbenzene with zinc ions in acetonitrile, enhancement of the fluorescence intensity was observed due to inhibition of intramolecular PET (photo-induced electron transfer) quenching, but no effect was found in aqueous media because the equilibrium laid to the free form of the ligands. In contrast, the addition of ATP disodium salt was effective to enhance the fluorescence intensity of the combination of the distyrylbenzne and Zn2+ in aqueous media. This assembly was applied to the peroxyoxalate chemiluminescence system and a significant increase in the intensity was observed, which provides a potential detection for ATP by chemiluminescence.

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

Science.gov (United States)

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

2012-12-01

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

The effects of low-intensity electromagnetic irradiation at the frequencies of 51.8 and 53 GHz and antibiotic ceftazidime on Lactobacillus acidophilus F0F1 ATP-ase activity

International Nuclear Information System (INIS)

Soghomonyan, D.R.

2013-01-01

The effects of low intensity electromagnetic irradiation (EMI) at the frequencies 51.8 and 53 GHz and antibiotic ceftazidime on N,N'-dicyclohexylcarbodiimide (DCCD), inhibited ATP-ase activity of Lactobacillus acidophilus membrane vesicles were investigated. It was shown that both frequencies decreased the ATP-ase activity, moreover, ceftazidime increase the sensitivity of cells to DCCD, inhibitor of the F 0 F 1 -ATP-ase. EMI combined with ceftazidime and DCCD markedly decreased the ATPase activity. The F 0 F 1 -ATP-ase is suggested can be a target for the effects observed

Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

International Nuclear Information System (INIS)

Ryu, T.; Davis, J.M.; Schwartz, K.A.

1990-01-01

The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding

Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

Science.gov (United States)

Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

2014-11-14

SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. Copyright © 2014 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

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

1989-01-01

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

Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

Energy Technology Data Exchange (ETDEWEB)

Morgunova, Ekaterina [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden); Illarionov, Boris; Saller, Sabine [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Popov, Aleksander [European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble CEDEX 09 (France); Sambaiah, Thota [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Bacher, Adelbert [Chemistry Department, Technical University of Munich, 85747 Garching (Germany); Cushman, Mark [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Fischer, Markus [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Ladenstein, Rudolf, E-mail: rudolf.ladenstein@ki.se [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden)

2010-09-01

Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

Energy Technology Data Exchange (ETDEWEB)

Somerville, Chris R.; Scieble, Wolf

2000-10-11

Cellulose synthase ('CS'), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

Benzalacetone Synthase

Directory of Open Access Journals (Sweden)

Ikuro eAbe

2012-03-01

Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

DEFF Research Database (Denmark)

Krath, B N; Hove-Jensen, B

2001-01-01

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...... is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two...... in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4....

Duchenne muscular dystrophy: normal ATP turnover in cultured cells

International Nuclear Information System (INIS)

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

1986-01-01

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

Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase

Directory of Open Access Journals (Sweden)

Xiang Zhou

2014-07-01

Full Text Available A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS. Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS. The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.

Virtual Screening of Novel Glucosamine-6-Phosphate Synthase Inhibitors.

Science.gov (United States)

Lather, Amit; Sharma, Sunil; Khatkar, Anurag

2018-01-01

Infections caused by microorganisms are the major cause of death today. The tremendous and improper use of antimicrobial agents leads to antimicrobial resistance. Various currently available antimicrobial drugs are inadequate to control the infections and lead to various adverse drug reactions. Efforts based on computer-aided drug design (CADD) can excavate a large number of databases to generate new, potent hits and minimize the requirement of time as well as money for the discovery of newer antimicrobials. Pharmaceutical sciences also have made development with advances in drug designing concepts. The current research article focuses on the study of various G-6-P synthase inhibitors from literature cited molecular database. Docking analysis was conducted and ADMET data of various molecules was evaluated by Schrodinger Glide and PreADMET software, respectively. Here, the results presented efficacy of various inhibitors towards enzyme G-6-P synthase. Docking scores, binding energy and ADMET data of various molecules showed good inhibitory potential toward G-6-P synthase as compared to standard antibiotics. This novel antimicrobial drug target G-6-P synthase has not so extensively been explored for its application in antimicrobial therapy, so the work done so far proved highly essential. This article has helped the drug researchers and scientists to intensively explore about this wonderful antimicrobial drug target. The Schrodinger, Inc. (New York, USA) software was utilized to carry out the computational calculations and docking studies. The hardware configuration was Intel® core (TM) i5-4210U CPU @ 2.40GHz, RAM memory 4.0 GB under 64-bit window operating system. The ADMET data was calculated by using the PreADMET tool (PreADMET ver. 2.0). All the computational work was completed in the Laboratory for Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, Rohtak, INDIA. Molecular docking studies were carried out to identify the binding

A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

Science.gov (United States)

Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

2008-10-01

Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

Science.gov (United States)

Zheng, Desen; Burr, Thomas J

2016-02-01

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae.

Science.gov (United States)

Chen, Xujun; Chen, Hao; Yuan, Joshua S; Köllner, Tobias G; Chen, Yuying; Guo, Yufen; Zhuang, Xiaofeng; Chen, Xinlu; Zhang, Yong-Jun; Fu, Jianyu; Nebenführ, Andreas; Guo, Zejian; Chen, Feng

2018-03-06

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up-regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Ketamine up-regulates a cluster of intronic miRNAs within the serotonin receptor 2C gene by inhibiting glycogen synthase kinase-3.

Science.gov (United States)

Grieco, Steven F; Velmeshev, Dmitry; Magistri, Marco; Eldar-Finkelman, Hagit; Faghihi, Mohammad A; Jope, Richard S; Beurel, Eleonore

2017-09-01

We examined mechanisms that contribute to the rapid antidepressant effect of ketamine in mice that is dependent on glycogen synthase kinase-3 (GSK3) inhibition. We measured serotonergic (5HT)-2C-receptor (5HTR2C) cluster microRNA (miRNA) levels in mouse hippocampus after administering an antidepressant dose of ketamine (10 mg/kg) in wild-type and GSK3 knockin mice, after GSK3 inhibition with L803-mts, and in learned helpless mice. Ketamine up-regulated cluster miRNAs 448-3p, 764-5p, 1264-3p, 1298-5p and 1912-3p (2- to 11-fold). This up-regulation was abolished in GSK3 knockin mice that express mutant constitutively active GSK3. The GSK3 specific inhibitor L803-mts was antidepressant in the learned helplessness and novelty suppressed feeding depression-like behaviours and up-regulated the 5HTR2C miRNA cluster in mouse hippocampus. After administration of the learned helplessness paradigm mice were divided into cohorts that were resilient (non-depressed) or were susceptible (depressed) to learned helplessness. The resilient, but not depressed, mice displayed increased hippocampal levels of miRNAs 448-3p and 1264-3p. Administration of an antagonist to miRNA 448-3p diminished the antidepressant effect of ketamine in the learned helplessness paradigm, indicating that up-regulation of miRNA 448-3p provides an antidepressant action. These findings identify a new outcome of GSK3 inhibition by ketamine that may contribute to antidepressant effects.

Complete inhibition of creatine kinase in isolated perfused rat hearts

International Nuclear Information System (INIS)

Fossel, E.T.; Hoefeler, H.

1987-01-01

Transient exposure of an isolated isovolumic perfused rat heart to low concentrations (0.5 mM) of perfusate-born iodoacetamide resulted in complete inhibition of creatine kinase and partial inhibition of glyceraldehyde-3-phosphate dehydrogenase in the heart. At low levels of developed pressure, hearts maintained mechanical function, ATP, and creatine phosphate levels at control values. However, iodoacetamide-inhibited hearts were unable to maintain control values of end diastolic pressure or peak systolic pressure as work load increased. Global ischemia resulted in loss of all ATP without loss of creatine phosphate, indicating lack of active creatine kinase. These results indicate that isovolumic perfused rat hearts are able to maintain normal function and normal levels of high-energy phosphates without active creatine kinase at low levels of developed pressure. 31 P-NMR of the heart was carried out

A taste for ATP: neurotransmission in taste buds

Science.gov (United States)

Kinnamon, Sue C.; Finger, Thomas E.

2013-01-01

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

A taste for ATP: neurotransmission in taste buds

Directory of Open Access Journals (Sweden)

Thomas E. Finger

2013-12-01

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

Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells.

Directory of Open Access Journals (Sweden)

Ayako Nakano

Full Text Available Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2, KG-1 (ABCB1 and HepG2 cells (ABCB1 and ABCG2. Interestingly, although side population (SP cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

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.

Packed red blood cells are an abundant and proximate potential source of nitric oxide synthase inhibition.

Directory of Open Access Journals (Sweden)

Charles F Zwemer

Full Text Available We determined, for packed red blood cells (PRBC and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS inhibitors asymmetric dimethylarginine (ADMA and monomethylarginine (LNMMA.ADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined.We measured total (free and protein incorporated ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis.In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM and LNMMA (58.9 ± 28.9 μM that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma.The compelling physiological ramifications are that regardless of storage age, 1 PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2 PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon

Optimisation of ATP determination in drinking water

DEFF Research Database (Denmark)

Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1991-01-01

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

Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.

Science.gov (United States)

Liu, Ying; Geng, Yue-Hang; Yang, Hui; Yang, Han; Zhou, Yan-Ting; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

2018-05-04

Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis. Copyright © 2018. Published by Elsevier B.V.

Dynamic changes in cytosolic ATP levels in cultured glutamatergic neurons during NMDA-induced synaptic activity supported by glucose or lactate

DEFF Research Database (Denmark)

Lange, Sofie Cecilie; Winkler, Ulrike; Andresen, Lars

2015-01-01

is supported equally well by both glucose and lactate, and that a pulse of NMDA causes accumulation of Ca(2+) in the mitochondrial matrix. In summary, we have shown that ATP homeostasis during neurotransmission activity in cultured neurons is supported by both glucose and lactate. However, ATP homeostasis...... biosensor Ateam1.03YEMK. While inducing synaptic activity by subjecting cultured neurons to two 30 s pulses of NMDA (30 µM) with a 4 min interval, changes in relative ATP levels were measured in the presence of lactate (1 mM), glucose (2.5 mM) or the combination of the two. ATP levels reversibly declined...... in the presence of glucose following the 2nd pulse of NMDA (approx. 10 vs. 20 %). Further, cytosolic Ca(2+) homeostasis during NMDA-induced synaptic transmission is partially inhibited by verapamil indicating that voltage-gated Ca(2+) channels are activated. Lastly, we showed that cytosolic Ca(2+) homeostasis...

Fatty Acid Synthase Activity as a Target for c-Met Driven Prostate Cancer

Science.gov (United States)

2013-07-01

cancer potentially due to increased fecal fat excretion. In addition, several families of plant-derived flavonoid compounds including...Apoptosis by Flavonoids Is Associated with Their Ability to Inhibit Fatty Acid Synthase Activity. J. Biol. Chem., 2005. 280(7): p. 5636-5645. 156... flavonoids , represent a source of relatively nontoxic, orally available and affordable compounds that are known to affect a number of different

Identification of potential leads against 4-hydroxytetrahydrodipicolinate synthase from Mycobacterium tuberculosis

OpenAIRE

Rehman, Ajijur; Akhtar, Salman; Siddiqui, Mohd Haris; Sayeed, Usman; Ahmad, Syed Sayeed; Arif, Jamal M.; Khan, M. Kalim A.

2016-01-01

4-hydroxy-tetrahydrodipicolinate synthase (DHDPS) is an important enzyme needed for the biosynthesis of lysine and many more key metabolites in Mycobacterium tuberculosis (Mtb). Inhibition of DHDPS is supposed to a promising therapeutic target due to its specific role in sporulation, cross-linking of the peptidiglycan polymers and biosynthesis of amino acids. In this work, a known inhibitor-based similarity search was carried out against a natural products database (Super Natural II) towards ...

wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs.

Science.gov (United States)

Lentz, Christian S; Halls, Victoria S; Hannam, Jeffrey S; Strassel, Silke; Lawrence, Sarah H; Jaffe, Eileen K; Famulok, Michael; Hoerauf, Achim; Pfarr, Kenneth M

2014-03-27

The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg(2+), or K(+) stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design of tailored PBGS modulators for potential application as antimicrobial agents, herbicides, or drugs for porphyric disorders.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions.

Science.gov (United States)

Harris, Kimberly A; Bobay, Benjamin G; Sarachan, Kathryn L; Sims, Alexis F; Bilbille, Yann; Deutsch, Christopher; Iwata-Reuyl, Dirk; Agris, Paul F

2015-08-14

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial ROS Production Protects the Intestine from Inflammation through Functional M2 Macrophage Polarization

Directory of Open Access Journals (Sweden)

Laura Formentini

2017-05-01

Full Text Available Mitochondria are signaling hubs in cellular physiology that play a role in inflammatory diseases. We found that partial inhibition of the mitochondrial ATP synthase in the intestine of transgenic mice triggers an anti-inflammatory response through NFκB activation mediated by mitochondrial mtROS. This shielding phenotype is revealed when mice are challenged by DSS-induced colitis, which, in control animals, triggers inflammation, recruitment of M1 pro-inflammatory macrophages, and the activation of the pro-oncogenic STAT3 and Akt/mTOR pathways. In contrast, transgenic mice can polarize macrophages to the M2 anti-inflammatory phenotype. Using the mitochondria-targeted antioxidant MitoQ to quench mtROS in vivo, we observe decreased NFκB activation, preventing its cellular protective effects. These findings stress the relevance of mitochondrial signaling to the innate immune system and emphasize the potential role of the ATP synthase as a therapeutic target in inflammatory and other related diseases.

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

Science.gov (United States)

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

2018-04-27

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

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

Science.gov (United States)

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

2013-08-01

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

NCBI nr-aa BLAST: CBRC-BTAU-01-1564 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-BTAU-01-1564 ref|YP_001382287.1| ATP synthase F0 subunit 6 [Argopecten irradia...ns] gb|ABS17670.1| ATP synthase F0 subunit 6 [Argopecten irradians] YP_001382287.1 1.1 24% ...

NCBI nr-aa BLAST: CBRC-XTRO-01-0090 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-XTRO-01-0090 ref|YP_001382287.1| ATP synthase F0 subunit 6 [Argopecten irradia...ns] gb|ABS17670.1| ATP synthase F0 subunit 6 [Argopecten irradians] YP_001382287.1 0.13 26% ...

NCBI nr-aa BLAST: CBRC-FCAT-01-0220 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-FCAT-01-0220 ref|YP_026082.1| ATP synthase F0 subunit 6 [Steinernema carpocaps...ae] gb|AAT00525.1| ATP synthase F0 subunit 6 [Steinernema carpocapsae] YP_026082.1 0.004 23% ...

NCBI nr-aa BLAST: CBRC-FCAT-01-0804 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-FCAT-01-0804 ref|YP_073313.1| ATP synthase F0 subunit 6 [Neomaskellia andropog...onis] gb|AAS75439.1| ATP synthase F0 subunit 6 [Neomaskellia andropogonis] YP_073313.1 1.8 24% ...

NCBI nr-aa BLAST: CBRC-DNOV-01-1349 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DNOV-01-1349 ref|YP_073313.1| ATP synthase F0 subunit 6 [Neomaskellia andropog...onis] gb|AAS75439.1| ATP synthase F0 subunit 6 [Neomaskellia andropogonis] YP_073313.1 1.8 25% ...

NCBI nr-aa BLAST: CBRC-DNOV-01-2699 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DNOV-01-2699 ref|YP_073313.1| ATP synthase F0 subunit 6 [Neomaskellia andropog...onis] gb|AAS75439.1| ATP synthase F0 subunit 6 [Neomaskellia andropogonis] YP_073313.1 0.055 30% ...

NCBI nr-aa BLAST: CBRC-OCUN-01-0782 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-OCUN-01-0782 ref|YP_073313.1| ATP synthase F0 subunit 6 [Neomaskellia andropog...onis] gb|AAS75439.1| ATP synthase F0 subunit 6 [Neomaskellia andropogonis] YP_073313.1 1.9 26% ...

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

Science.gov (United States)

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

2013-10-08

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

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

Science.gov (United States)

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

2013-01-01

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

Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

International Nuclear Information System (INIS)

Mistafa, Oras; Hoegberg, Johan; Stenius, Ulla

2008-01-01

Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells

Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Mistafa, Oras; Hoegberg, Johan [Institute of Environmental Medicine, Karolinska Institutet, Box 210, 17177 Stockholm (Sweden); Stenius, Ulla [Institute of Environmental Medicine, Karolinska Institutet, Box 210, 17177 Stockholm (Sweden)

2008-01-04

Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells.

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

Science.gov (United States)

Michailova, A.; McCulloch, A.

2001-01-01

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

Activity of mevalonate pathway inhibitors against breast and ovarian cancers in the ATP-based tumour chemosensitivity assay

International Nuclear Information System (INIS)

Knight, Louise A; Kurbacher, Christian M; Glaysher, Sharon; Fernando, Augusta; Reichelt, Ralf; Dexel, Susanne; Reinhold, Uwe; Cree, Ian A

2009-01-01

Previous data suggest that lipophilic statins such as fluvastatin and N-bisphosphonates such as zoledronic acid, both inhibitors of the mevalonate metabolic pathway, have anti-cancer effects in vitro and in patients. We have examined the effect of fluvastatin alone and in combination with zoledronic acid in the ATP-based tumour chemosensitivity assay (ATP-TCA) for effects on breast and ovarian cancer tumour-derived cells. Both zoledronic acid and fluvastatin showed activity in the ATP-TCA against breast and ovarian cancer, though fluvastatin alone was less active, particularly against breast cancer. The combination of zoledronic acid and fluvastatin was more active than either single agent in the ATP-TCA with some synergy against breast and ovarian cancer tumour-derived cells. Sequential drug experiments showed that pre-treatment of ovarian tumour cells with fluvastatin resulted in decreased sensitivity to zoledronic acid. Addition of mevalonate pathway components with zoledronic acid with or without fluvastatin showed little effect, while mevalonate did reduced inhibition due to fluvastatin. These data suggest that the combination of zoledronic acid and fluvastatin may have activity against breast and ovarian cancer based on direct anti-cancer cell effects. A clinical trial to test this is in preparation

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

International Nuclear Information System (INIS)

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

1998-01-01

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

NCBI nr-aa BLAST: CBRC-MDOM-04-0135 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-MDOM-04-0135 ref|YP_073313.1| ATP synthase F0 subunit 6 [Neomaskellia andropog...onis] gb|AAS75439.1| ATP synthase F0 subunit 6 [Neomaskellia andropogonis] YP_073313.1 3e-04 28% ...

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

Energy Technology Data Exchange (ETDEWEB)

Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

2017-07-03

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

International Nuclear Information System (INIS)

Grum, Daniel; Boom, Johannes van den; Neumann, Daniel; Matena, Anja; Link, Nina M.; Mueller, Jonathan W.

2010-01-01

3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

NCBI nr-aa BLAST: CBRC-TTRU-01-1294 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-TTRU-01-1294 ref|NP_051157.1| ATP synthase F0 subunit 6 [Cafeteria roenbergens...is] gb|AAF05808.1|AF193903_31 ATP synthase F0 subunit 6 [Cafeteria roenbergensis] NP_051157.1 0.057 29% ...

NCBI nr-aa BLAST: CBRC-DNOV-01-0972 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DNOV-01-0972 ref|NP_051157.1| ATP synthase F0 subunit 6 [Cafeteria roenbergens...is] gb|AAF05808.1|AF193903_31 ATP synthase F0 subunit 6 [Cafeteria roenbergensis] NP_051157.1 0.001 29% ...

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

Science.gov (United States)

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

2011-10-01

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

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

Science.gov (United States)

Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

2014-05-01

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

ATP-consuming and ATP-generating enzymes secreted by pancreas

DEFF Research Database (Denmark)

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

2006-01-01

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

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

DEFF Research Database (Denmark)

Koul, A.; Vranckx, L.; Dhar, N.

2014-01-01

Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using...... microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional...... and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non...

Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains