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Sample records for monophosphate kinase mrna

  1. Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney B. (Pullman, WA); Lange, Bernd M. (Pullman, WA)

    2001-01-01

    A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase 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 isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

  2. Structure of Staphylococcus aureus cytidine monophosphate kinase in complex with cytidine 5'-monophosphate.

    Science.gov (United States)

    Dhaliwal, Balvinder; Ren, Jingshan; Lockyer, Michael; Charles, Ian; Hawkins, Alastair R; Stammers, David K

    2006-08-01

    The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 5'-monophosphate (CMP) has been determined at 2.3 angstroms resolution. The active site reveals novel features when compared with two orthologues of known structure. Compared with the Streptococcus pneumoniae CMK solution structure of the enzyme alone, S. aureus CMK adopts a more closed conformation, with the NMP-binding domain rotating by approximately 16 degrees towards the central pocket of the molecule, thereby assembling the active site. Comparing Escherichia coli and S. aureus CMK-CMP complex structures reveals differences within the active site, including a previously unreported indirect interaction of CMP with Asp33, the replacement of a serine residue involved in the binding of CDP by Ala12 in S. aureus CMK and an additional sulfate ion in the E. coli CMK active site. The detailed understanding of the stereochemistry of CMP binding to CMK will assist in the design of novel inhibitors of the enzyme. Inhibitors are required to treat the widespread hospital infection methicillin-resistant S. aureus (MRSA), currently a major public health concern.

  3. CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

    Science.gov (United States)

    Bucurenci, N; Sakamoto, H; Briozzo, P; Palibroda, N; Serina, L; Sarfati, R S; Labesse, G; Briand, G; Danchin, A; Bărzu, O; Gilles, A M

    1996-02-02

    CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

  4. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

    Science.gov (United States)

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  5. Protective effects of inhibition of adenosine monophosphate activated protein kinase activity against cerebral ischemia-reperfusion injury in mice

    Institute of Scientific and Technical Information of China (English)

    补娟

    2013-01-01

    Objective To observe the effect of inhibition of adenosine monophosphate activated protein kinase (AMPK) on shape,function and inflammatory factor of microglia for mice after cerebral ischemia-reperfusion

  6. Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds.

    Science.gov (United States)

    Marín-Aguilar, Fabiola; Pavillard, Luis E; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D

    2017-01-29

    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases.

  7. Metabolic syndrome: adenosine monophosphate-activated protein kinase and malonyl coenzyme A.

    Science.gov (United States)

    Ruderman, Neil B; Saha, Asish K

    2006-02-01

    The metabolic syndrome can be defined as a state of metabolic dysregulation characterized by insulin resistance, central obesity, and a predisposition to type 2 diabetes, dyslipidemia, premature atherosclerosis, and other diseases. An increasing body of evidence has linked the metabolic syndrome to abnormalities in lipid metabolism that ultimately lead to cellular dysfunction. We review here the hypothesis that, in many instances, the cause of these lipid abnormalities could be a dysregulation of the adenosine monophosphate-activated protein kinase (AMPK)/malonyl coenzyme A (CoA) fuel-sensing and signaling mechanism. Such dysregulation could be reflected by isolated increases in malonyl CoA or by concurrent changes in malonyl CoA and AMPK, both of which would alter intracellular fatty acid partitioning. The possibility is also raised that pharmacological agents and other factors that activate AMPK and/or decrease malonyl CoA could be therapeutic targets.

  8. Kinetic mechanism and energetics of binding of phosphoryl group acceptors to Mycobacterium tuberculosis cytidine monophosphate kinase.

    Science.gov (United States)

    Jaskulski, Léia; Rosado, Leonardo A; Rostirolla, Diana C; Timmers, Luis F S M; de Souza, Osmar N; Santos, Diogenes S; Basso, Luiz A

    2013-08-01

    Cytidine monophosphate kinase from Mycobacterium tuberculosis (MtCMK) likely plays a role in supplying precursors for nucleic acid synthesis. MtCMK catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Initial velocity studies and Isothermal titration calorimetry (ITC) measurements showed that MtCMK follows a random-order mechanism of substrate (CMP and ATP) binding, and an ordered mechanism for product release, in which ADP is released first followed by CDP. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2'-OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons between CMP/dCMP kinases and UMP/CMP ones.

  9. 5'-adenosine monophosphate-activated protein kinase and the metabolic syndrome.

    Science.gov (United States)

    Mor, Vijay; Unnikrishnan, M K

    2011-09-01

    Lifestyle changes such as physical inactivity combined with calorie-rich, low-fibre diets have triggered an explosive surge in metabolic syndrome, outlined as a cluster of heart attack risk factors such as insulin resistance, raised fasting plasma glucose, abdominal obesity, high cholesterol and high blood pressure. By acting as a master-switch of energy homeostasis and associated pathophysiological phenomena, 5'-adenosine monophosphate-activated protein kinase (AMPK) appears to orchestrate the adaptive physiology of energy deficit, suggesting that the sedentary modern human could be suffering from chronic suboptimal AMPK activation. Addressing individual targets with potent ligands with high specificity may be inappropriate (it has not yielded any molecule superior to the sixty year old metformin) because this strategy cannot address a cluster of interrelated pathologies. However, spices, dietary supplements and nutraceuticals attenuate the multiple symptoms of metabolic syndrome in a collective and perhaps more holistic fashion with fewer adverse events. Natural selection could have favoured races that developed a taste for spices and dietary supplements, most of which are not only antioxidants but also activators of AMPK. The review will outline the various biochemical mechanisms and pathophysiological consequences of AMPK activation involving the cluster of symptoms that embrace metabolic syndrome and beyond. Recent advances that integrate energy homeostasis with a number of overarching metabolic pathways and physiological phenomena, including inflammatory conditions, cell growth and development, malignancy, life span, and even extending into environmental millieu, as in obesity mediated by gut microflora and others will also be outlined.

  10. Effects of adenosine 5’monophosphate-activated protein kinase on europrotection induced by ischemic preconditioning

    Directory of Open Access Journals (Sweden)

    Yuan-ru-hua TIAN

    2015-06-01

    Full Text Available Objective To investigate the effects of adenosine 5'-monophosphate-activated protein kinase (AMPK and phosphated AMPK (pAMPK signals in ischemic preconditioning (IPC, and the effect of pharmacological intervention of AMPK on infarct size of the brain. Methods A brief (3min middle cerebral artery occlusion (MCAO was employed to induce IPC in male rat, and another 90-min MCAO was performed 4 or 72h later. The levels of AMPK and pAMPK were assessed after IPC. A pharmacological activator metformin, or inhibitor compound C of AMPK, was used to analyze the correlation of IPC to AMPK signaling in MCAO rats. Results The infarct size of total cerebral hemisphere and cortex was significantly decreased in MCAO animals by IPC for 72h (P0.05, n=6. The AMPK activator metformin can significantly reverse the protective effect of IPC (P<0.05, n=6. Conclusions The signals of AMPK and pAMPK play an important role in neuroprotective effect of IPC on cerebral ischemic injury. The neuroprotective effect of IPC may be associated with the down-regulation of pAMPK. DOI: 10.11855/j.issn.0577-7402.2015.05.07

  11. Cyclic Nucleotide-Dependent Protein Kinases, IV. Widespread Occurrence of Adenosine 3′,5′-monophosphate-dependent Protein Kinase in Various Tissues and Phyla of the Animal Kingdom

    National Research Council Canada - National Science Library

    J. F. Kuo; Paul Greengard

    1969-01-01

    Adenosine 3 ,5 -monophosphate-dependent protein kinase activity was found in about thirty sources including many mammalian tissues as well as species representative of eight different invertebrate phyla...

  12. Adenosine monophosphate-activated protein kinase from the mud crab, Scylla paramamosain: cDNA cloning and profiles under cold stress

    Indian Academy of Sciences (India)

    CHENCUI HUANG; KUN YU; HUIYANG HUANG; HAIHUI YE

    2016-12-01

    Adenosine monophosphate-activated protein kinase (AMPK), an important energy sensor, is crucial for organism survival under adverse conditions. In this study, the roles of this gene under cold stress in a warm-water mud crab, Scylla paramamosain was investigated. The full-length cDNA (SpAMPK) was 1884 bp and its open reading frame of 1566 bp was isolated and characterized. The expressions of SpAMPK detected by quantitative real-time PCR (qRT-PCR) in various tissues revealed that the highest expression was in the hepatopancreas. The profiles of SpAMPK gene in the hepatopancreas, chela muscleand gill were detected when the subadult crabs were exposed to the four temperature conditions of 10, 15, 20 and 25◦C. The results showed that the expression patterns of SpAMPK mRNA in the three tissues were significantly higher when crabs were exposed to 15◦C than the other three temperature treatments, while at 10◦C treatment, the SpAMPK mRNA was lowestamong the four temperature treatments. These findings suggested that the high expression of SpAMPK mRNA might initiate ATP-producing pathways to generate energy to cope with cold stress at 15◦C treatment, which was slightly below the range of optimum temperatures; while treatment at 10◦C, far lower than optima, the low expression of SpAMPK mRNA could reduce the energy expenditure and thus induce the crabs into cold anesthesia. The results of SpAMPK in this study might contribute to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain.

  13. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    Science.gov (United States)

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2016-12-01

    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca(2+) influx via a mechanosensitive L-type Ca(2+) channel, which subsequently raises intracellular Ca(2+) and activates AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca(2+)-channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  14. Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture : Response to Cell Wall-Degrading Enzymes.

    Science.gov (United States)

    Tan, Z; Boss, W F

    1992-12-01

    Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation.

  15. Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture 1

    Science.gov (United States)

    Tan, Zheng; Boss, Wendy F.

    1992-01-01

    Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation. Images Figure 2 PMID:16653250

  16. Growth inhibition of human gastric adenocarcinoma cells in vitro by STO-609 is independent of calcium/calmodulin-dependent protein kinase kinase-beta and adenosine monophosphate-activated protein kinase.

    Science.gov (United States)

    Ma, Zhiming; Wen, Dacheng; Wang, Xudong; Yang, Longfei; Liu, Tianzhou; Liu, Jingjing; Zhu, Jiaming; Fang, Xuedong

    2016-01-01

    Adenosine monophosphate (AMP)-activated protein kinase is a recently identified downstream target of calcium/calmodulin-dependent protein kinase kinase-beta, and is involved in the regulation of cell metabolism and cell proliferation. STO-609 is a selective antagonist of calcium/calmodulin-dependent protein kinase kinase-beta. In the present study, we found that STO-609 suppressed AMP-activated protein kinase activity, reduced expression of Akt and ERK, and increased cell apoptosis in SNU-1 and N87 cells but not normal gastric epithelial cells (CCL-241). Interestingly, we found such effects of STO-609 on gastric cancer cells were not affected after the knock-down of CaMKK-β and AMPK. In conclusion, STO-609 is an effective cytotoxic agent for gastric adenocarcinoma in vivo.

  17. Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

    Science.gov (United States)

    Mehta, Suresh L; Mendelev, Natalia; Kumari, Santosh; Andy Li, P

    2013-03-01

    Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and

  18. Computer-assisted combinatorial design of bicyclic thymidine analogs as inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase

    Science.gov (United States)

    Frecer, Vladimir; Seneci, Pierfausto; Miertus, Stanislav

    2011-01-01

    Thymidine monophosphate kinase (TMPKmt) is an essential enzyme for nucleotide metabolism in Mycobacterium tuberculosis, and thus an attractive target for novel antituberculosis agents. In this work, we have explored the chemical space around the 2',3'-bicyclic thymidine nucleus by designing and in silico screening of a virtual focused library selected via structure based methods to identify more potent analogs endowed with favorable ADME-related properties. In all the library members we have exchanged the ribose ring of the template with a cyclopentane moiety that is less prone to enzymatic degradation. In addition, we have replaced the six-membered 2',3'-ring by a number of five-membered and six-membered heterocyclic rings containing alternative proton donor and acceptor groups, to exploit the interaction with the carboxylate groups of Asp9 and Asp163 as well as with several cationic residues present in the vicinity of the TMPKmt binding site. The three-dimensional structure of the TMPKmt complexed with 5-hydroxymethyl-dUMP, an analog of dTMP, was employed to develop a QSAR model, to parameterize a scoring function specific for the TMPKmt target and to select analogues which display the highest predicted binding to the target. As a result, we identified a small highly focused combinatorial subset of bicyclic thymidine analogues as virtual hits that are predicted to inhibit the mycobacterial TMPK in the submicromolar concentration range and to display favorable ADME-related properties.

  19. Methylene blue induces macroautophagy through 5′ adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation

    Science.gov (United States)

    Xie, Luokun; Li, Wenjun; Winters, Ali; Yuan, Fang; Jin, Kunlin; Yang, Shaohua

    2013-01-01

    Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with in vitro data, macroautophagy was induced in the cortex and hippocampus of mouse brains treated with methylene blue. Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling. PMID:23653592

  20. Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation.

    Science.gov (United States)

    Xie, Luokun; Li, Wenjun; Winters, Ali; Yuan, Fang; Jin, Kunlin; Yang, Shaohua

    2013-01-01

    Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with in vitro data, macroautophagy was induced in the cortex and hippocampus of mouse brains treated with methylene blue. Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling.

  1. Structure-based in-silico rational design of a selective peptide inhibitor for thymidine monophosphate kinase of mycobacterium tuberculosis.

    Science.gov (United States)

    Kumar, Manoj; Sharma, Sujata; Srinivasan, Alagiri; Singh, Tej P; Kaur, Punit

    2011-05-01

    Tuberculosis still remains one of the most deadly infectious diseases. The emergence of drug resistant strains has fuelled the quest for novel drugs and drug targets for its successful treatment. Thymidine monophosphate kinase (TMPK) lies at the point where the salvage and de novo synthetic pathways meet in nucleotide synthesis. TMPK in M.tb has emerged as an attractive drug target since blocking it will affect both the pathways involved in the thymidine triphosphate synthesis. Moreover, the unique differences at the active site of TMPK enzyme in M.tb and humans can be exploited for the development of ideal drug candidates. Based on a detailed evaluation of known inhibitors and available three-dimensional structures of TMPK, several peptidic inhibitors were designed. In silico docking and selectivity analysis of these inhibitors with TMPK from M.tb and human was carried out to examine their differential binding at the active site. The designed tripeptide, Trp-Pro-Asp, was found to be most selective for M.tb. The ADMET analysis of this peptide indicated that it is likely to be a drug candidate. The tripeptide so designed is a suitable lead molecule for the development of novel TMPK inhibitors as anti-tubercular drugs.

  2. Synthèse et fonctionnalisation de 2-thiohydantoïnes : interaction et inhibition des nucléosides monophosphate kinases

    OpenAIRE

    Gosling, Sandrine

    2011-01-01

    New therapeutical compounds determination requires the formation of a library of molecules and their screening on specific biological targets. The aim of this project was to design new inhibitors targeting nucléoside monophosphate kinases (NMPK) based on in situ dynamic combinatorial chemistry. These molecules were synthesized by ligation between analogues of phosphate acceptors and donors on which reactive functions were introduced. The topic of this PhD was to develop the ATP mimetics using...

  3. The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

    Directory of Open Access Journals (Sweden)

    Galo L. Mejia

    2016-08-01

    Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

  4. Hepatitis C virus core protein induces energy metabolism disorders of hepatocytes by down-regulation of silent mating type information regulation 2 homolog-1 and adenosine monophosphate-acti vated protein kinase signaling pathway

    Institute of Scientific and Technical Information of China (English)

    于建武

    2013-01-01

    Objective To study the role of silent mating type information regulation2homotog-1(SIRT1)-adenosine monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway in hepatitis C virus core protein(HCV-core)induced energy metabolism disorders

  5. Role of adenosine 5'-monophosphate-activated protein kinase in interleukin-6 release from isolated mouse skeletal muscle

    DEFF Research Database (Denmark)

    Glund, Stephan; Treebak, Jonas Thue; Long, Yun Chau;

    2009-01-01

    IL-6 is released from skeletal muscle during exercise and has consequently been implicated to mediate beneficial effects on whole-body metabolism. Using 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), a pharmacological activator of 5'-AMP-activated protein kinase (AMPK), we tested...... the hypothesis that AMPK modulates IL-6 release from isolated muscle. Skeletal muscle from AMPKalpha2 kinase-dead transgenic, AMPKalpha1 knockout (KO) and AMPKgamma3 KO mice and respective wild-type littermates was incubated in vitro, in the absence or presence of 2 mmol/liter AICAR. Skeletal muscle from wild......-type mice was also incubated with the AMPK activator A-769662. Incubation of mouse glycolytic extensor digitorum longus and oxidative soleus muscle for 2 h was associated with profound IL-6 mRNA production and protein release, which was suppressed by AICAR (P

  6. Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-fang; ZHANG Jin-ying; LI Ling; ZHAO Xiao-yan

    2011-01-01

    Background Metformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin.Methods Cardiomyocytes were incubated in the presence of 100 umol/L. H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide (AICAR) (500 umol/L), an adenosine monophophate (AMP)-activated protein kinase (AMPK) agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C (an AMPK antagonist, 20 umol/L) for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase (eNOS), and transforming growth factor (TGF)-β1 were analyzed using immunblotting.Results Metformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α.Conclusions Metformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.

  7. Role of activation of 5'-adenosine monophosphate-activated protein kinase in gastric ulcer healing in diabetic rats.

    Science.gov (United States)

    Baraka, Azza M; Deif, Maha M

    2011-01-01

    The potential utility of 5'-adenosine monophosphate-activated protein kinase (AMPK)-activating agents, such as metformin, in inducing angiogenesis, could be a promising approach to promote healing of gastric ulcers complicated by diabetes mellitus. The aim of the present study was to assess the effect of a drug that activates AMPK, namely metformin, in gastric ulcer healing in streptozotocin-induced diabetic rats. Forty male Wistar albino rats were made diabetic by intraperitoneal (i.p.) streptozotocin injection and 10 rats were injected i.p. by a single dose of physiological saline. Six weeks following streptozotocin or saline injection, gastric ulcers were induced by serosal application of acetic acid. Three days after acetic acid application, rats were divided into group 1 (nondiabetic control), group 2 (streptozotocin-injected rats), groups 3-5 (streptozotocin-injected rats treated with metformin or metformin and an inhibitor of AMPK, namely compound C or pioglitazone) for 7 days following acetic acid application. Administration of metformin, but not pioglitazone, resulted in a significant decrease in the gastric ulcer area, a significant increase in epithelial regeneration assessed histologically, a significant increase in the number of microvessels in the ulcer margin, a significant increase in gastric vascular endothelial growth factor concentration and gastric von Willebrand factor as well as a significant increase in gastric phospho-AMPK. Compound C, an inhibitor of AMPK, blocked metformin-induced changes in assessed parameters suggesting that the effect of metformin was mediated mainly through activation of AMPK. Our results suggest the feasibility of a novel treatment strategy, namely drugs activating AMPK, for patients in whom impairment of ulcer healing constitutes a secondary complication of diabetes mellitus. Copyright © 2011 S. Karger AG, Basel.

  8. Changes in adenosine 5'-monophosphate-activated protein kinase as a mechanism of visceral obesity in Cushing's syndrome.

    Science.gov (United States)

    Kola, Blerina; Christ-Crain, Mirjam; Lolli, Francesca; Arnaldi, Giorgio; Giacchetti, Gilberta; Boscaro, Marco; Grossman, Ashley B; Korbonits, Márta

    2008-12-01

    Features of the metabolic syndrome such as central obesity with insulin resistance and dyslipidemia are typical signs of Cushing's syndrome and common side effects of prolonged glucocorticoid treatment. AMP-activated protein kinase (AMPK), a key regulatory enzyme of lipid and carbohydrate metabolism as well as appetite, is involved in the development of the deleterious metabolic effects of excess glucocorticoids, but no data are available in humans. In the current study, we demonstrate the effect of high glucocorticoid levels on AMPK activity of human adipose tissue samples from patients with Cushing's syndrome. AMPK activity and mRNA expression of genes involved in lipid metabolism were assessed in visceral adipose tissue removed at abdominal surgery of 11 patients with Cushing's syndrome, nine sex-, age-, and weight-matched patients with adrenal incidentalomas, and in visceral adipose tissue from four patients with non-endocrine-related abdominal surgery. The patients with Cushing's syndrome exhibited a 70% lower AMPK activity in visceral adipose tissue as compared with both incidentalomas and control patients (P = 0.007 and P cortisol and with urinary free cortisol. Our data suggest that glucocorticoids inhibit AMPK activity in adipose tissue, suggesting a novel mechanism to explain the deposition of visceral adipose tissue and the consequent central obesity observed in patients with iatrogenic or endogenous Cushing's syndrome.

  9. Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-12-01

    Full Text Available Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

  10. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    Science.gov (United States)

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  11. Resistance artery creatine kinase mRNA and blood pressure in humans.

    Science.gov (United States)

    Karamat, Fares A; Oudman, Inge; Ris-Stalpers, Carrie; Afink, Gijs B; Keijser, Remco; Clark, Joseph F; van Montfrans, Gert A; Brewster, Lizzy M

    2014-01-01

    Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

  12. Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Handa, A.K.; Bressan, R.A.

    1980-03-01

    In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: (ATP) = (ATP)/sub 0/ e/sup -(cAMP)kt/. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.

  13. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie

    2011-01-01

    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.

  14. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    Science.gov (United States)

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver.

  15. Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

    2011-11-01

    Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

  16. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr...

  17. Nitration of Tyrosine 247 Inhibits Protein Kinase G-1α Activity by Attenuating Cyclic Guanosine Monophosphate Binding*

    Science.gov (United States)

    Aggarwal, Saurabh; Gross, Christine M.; Rafikov, Ruslan; Kumar, Sanjiv; Fineman, Jeffrey R.; Ludewig, Britta; Jonigk, Danny; Black, Stephen M.

    2014-01-01

    The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr247 is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr247 would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [3H]cGMP binding assay. Our study shows that the nitration of Tyr247 and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation. PMID:24469460

  18. Nitration of tyrosine 247 inhibits protein kinase G-1α activity by attenuating cyclic guanosine monophosphate binding.

    Science.gov (United States)

    Aggarwal, Saurabh; Gross, Christine M; Rafikov, Ruslan; Kumar, Sanjiv; Fineman, Jeffrey R; Ludewig, Britta; Jonigk, Danny; Black, Stephen M

    2014-03-14

    The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr(247) is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr(247) would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [(3)H]cGMP binding assay. Our study shows that the nitration of Tyr(247) and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation.

  19. Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    Yanling Sui; Zichun Zhao; Rong Liu; Bin Cai; Dongsheng Fan

    2014-01-01

    Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis-mutase 1 mutant (SOD1G93A) and wild-type (SOD1WT) mouse models were exposed to H2O2. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by lfow cytometry. Moreover, we evaluated the expression of the adenos-ine monophosphate-activated protein kinase (AMPK)α-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1WT cells, SOD1G93A embryonic neural stem cells were more likely to undergo H2O2-induced apoptosis. Phosphorylation of AMPKαin SOD1G93A cells was higher than that in SOD1WT cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKα. p53 protein levels were also correlated with AMPKαphosphorylation levels. Compound C, an inhibitor of AMPKα, attenuated the effects of H2O2. These results suggest that embryonic neural stem cells from SOD1G93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKα pathway.

  20. Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

    Science.gov (United States)

    Yerra, Veera Ganesh; Kumar, Ashutosh

    2017-04-01

    Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30 mg/kg, i.p) was administered to Sprague-Dawley rats (250-270 g) for 2 weeks after 6 weeks of streptozotocin (STZ) injection (55 mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25 mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250 mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

  1. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

    Institute of Scientific and Technical Information of China (English)

    Yu Mei; Wen-Jing Jia; Yu-Jia Chu; Hong-Wei Xue

    2012-01-01

    Phosphatidylinositol monophosphate 5-kinase(PIP5K)catalyzes the synthesis of PI-4,5-bisphosphate(PtdIns(4,5)P2)by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring,and is involved in regulating multiple developmental processes and stress responses.We here report on the functional characterization of Arabidopsis PIP5K2,which is expressed during lateral root initiation and elongation,and whose expression is enhanced by exogenous auxin.The knockout mutant pip5k2 shows reduced lateral root formation,which could be recovered with exogenous auxin,and interestingly,delayed root gravity response that could not be recovered with exogenous auxin.Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2.In addition,analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P2 reduction,which hence results in suppressed cycling of PIN proteins(PIN2 and 3),and delayed redistribution of PIN2 and auxin under gravistimulation in pipSk2 roots.On the contrary,PtdIns(4,5)P2 significantly enhanced the vesicle trafficking and cycling of PIN proteins.These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response,and reveal a critical role of PIP5K2/Ptdlns(4,5)P2 in root development through regulation of PIN proteins,providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response,and new insights into the control of polar auxin transport.

  2. Globular adiponectin protects human umbilical vein endothelial cells against apoptosis through adiponectin receptor 1/adenosine monophosphate-activated protein kinase pathway

    Institute of Scientific and Technical Information of China (English)

    ZHAO Hong-yu; ZHAO Min; YI Tong-ning; ZHANG Jin

    2011-01-01

    Background Endothelial dysfunction is a key event in the onset and progression of atherosclerosis in diabetic patients.Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. However, no study has addressed apoptosis in human umbilical vein endothelial cells (HUVECs) induced by an intermittent high-glucose media and its association with adiponectin receptor 1 (adipoR1), adipoR2, or adenosine monophosphate (AMP)-activated protein kinase (AMPK).Methods HUVECs were cultured in continuous normal glucose (5.5 mmol/L), continuous high glucose (25 mmol/L),alternating normal and high glucose and mannitol. In the alternating normal and high-glucose media, HUVECs were treated under different conditions. First, cells were transfected with the adipoR1-specific small-interfering RNA (siRNA)and then stimulated with globular adiponectin (gAD). Second, cells were cultured in both gAD and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Third, cells were cultured in the AMPK inhibitor adenine-9-β-D-arabino-furanoside (araA), gAD, and in AICAR.Results HUVEC apoptosis increased more significantly in an intermittent high-glucose medium than in a constant high-glucose medium. HUVEC apoptosis induced by an intermittent high-glucose medium was inhibited when the cells were pretreated with 3 μg/ml gAD, which rapidly activated AMPK and adipoR1 in HUVECs. However, adipoR2 was not activated.Conclusions We found that adipoR1, not adipoR2, is involved in mediating intermittent high-concentration glucoseevoked apoptosis in endothelial cells. gAD activated AMPK through adipoR1, leads to the partial inhibition of HUVEC apoptosis. A fluctuating glucose medium is more harmful than a constant high-glucose medium to endothelial cells.

  3. SIRT1/Adenosine Monophosphate-Activated Protein Kinase α Signaling Enhances Macrophage Polarization to an Anti-inflammatory Phenotype in Rheumatoid Arthritis.

    Science.gov (United States)

    Park, So Youn; Lee, Sung Won; Lee, Sang Yeob; Hong, Ki Whan; Bae, Sun Sik; Kim, Koanhoi; Kim, Chi Dae

    2017-01-01

    Macrophages are crucially involved in the pathogenesis of rheumatoid arthritis (RA). Macrophages of the M1 phenotype act as pro-inflammatory mediators in synovium, whereas those of the M2 phenotype suppress inflammation and promote tissue repair. SIRT1 is a class 3 histone deacetylase with anti-inflammatory characteristics. However, the role played by SIRT1 in macrophage polarization has not been defined in RA. We investigated whether SIRT1 exerts anti-inflammatory effects by modulating M1/M2 polarization in macrophages from RA patients. In this study, SIRT1 activation promoted the phosphorylation of an adenosine monophosphate-activated protein kinase (AMPK) α/acetyl-CoA carboxylase in macrophages exposed to interleukin (IL)-4, and that this resulted in the expressions of M2 genes, including MDC, FcεRII, MrC1, and IL-10, at high levels. Furthermore, these expressions were inhibited by sirtinol (an inhibitor of SIRT1) and compound C (an inhibitor of AMPK). Moreover, SIRT1 activation downregulated LPS/interferon γ-mediated NF-κB activity by inhibiting p65 acetylation and the expression of M1 genes, such as CCL2, iNOS, IL-12 p35, and IL-12 p40. Macrophages from SIRT1 transgenic (Tg)-mice exhibited enhanced polarization of M2 phenotype macrophages and reduced polarization of M1 phenotype macrophages. In line with these observations, SIRT1-Tg mice showed less histological signs of arthritis, that is, lower TNFα and IL-1β expressions and less severe arthritis in the knee joints, compared to wild-type mice. Taken together, the study shows activation of SIRT1/AMPKα signaling exerts anti-inflammatory activities by regulating M1/M2 polarization, and thereby reduces inflammatory responses in RA. Furthermore, it suggests that SIRT1 signaling be viewed as a therapeutic target in RA.

  4. A high isoflavone diet decreases 5' adenosine monophosphate-activated protein kinase activation and does not correct selenium-induced elevations in fasting blood glucose in mice.

    Science.gov (United States)

    Stallings, Michael T; Cardon, Brandon R; Hardman, Jeremy M; Bliss, Tyler A; Brunson, Scott E; Hart, Chris M; Swiss, Maria D; Hepworth, Squire D; Christensen, Merrill J; Hancock, Chad R

    2014-04-01

    Selenium (Se) has been implicated as a micronutrient that decreases adenosine monophosphate-activated protein kinase (AMPK) signaling and may increase diabetes risk by reducing insulin sensitivity. Soy isoflavones (IF) are estrogen-like compounds that have been shown to attenuate insulin resistance, hyperglycemia, adiposity, and increased AMPK activation. We hypothesized that a high IF (HIF) diet would prevent the poor metabolic profile associated with high Se intake. The purpose of this study was to examine changes in basal glucose metabolism and AMPK signaling in response to an HIF diet and/or supplemental Se in a mouse model. Male FVB mice were divided into groups receiving either a control diet with minimal IF (low IF) or an HIF diet. Each dietary group was further subdivided into groups receiving either water or Se at a dose of 3 mg Se/kg body weight daily, as Se-methylselenocysteine (SMSC). After 5 months, mice receiving SMSC had elevated fasting glucose (P < .05) and a tendency for glucose intolerance (P = .08). The increase in dietary IF did not result in improved fasting blood glucose. Interestingly, after 6 months, HIF-fed mice had decreased basal AMPK activation in liver and skeletal muscle tissue (P < .05). Basal glucose metabolism was changed by SMSC supplementation as evidenced by increased fasting blood glucose and glucose intolerance. High dietary IF levels did not protect against aberrant blood glucose. In FVB mice, decreased basal AMPK activation is not the mechanism through which Se exerts its effect. These results suggest that more research must be done to elucidate the role of Se and IF in glucose metabolism.

  5. Metformin inhibits nuclear factor-κB activation and inflammatory cytokines expression induced by high glucose via adenosine monophosphate-activated protein kinase activation in rat glomerular mesangial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Gu Junfei; Ye Shandong; Wang Shan; Sun Wenjia; Hu Yuanyuan

    2014-01-01

    Background The renoprotective mechanisms of adenosine monophosphate (AMP)-activated protein kinase (AMPK) agonist-metformin have not been stated clearly.We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB (NF-κB),monocyte chemoattractant protein-1 (MCP-1),intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-β1) induced by high glucose (HG) in cultured rat glomerular mesangial cells (MCs).Methods MCs were cultured in the medium with normal concentration glucose (group NG,5.6 mmol/L),high concentration glucose (group HG,25 mmol/L) and different concentrations of metformin (group M1,M2,M3).After 48-hour exposure,the supernatants and MCs were collected.The expression of NF-κB,MCP-1,ICAM-1,and TGF-β1 mRNA was analyzed by real time polymerase chain reaction.Westem blotting was used to detect the expression of AMPK,phospho-Thr-172 AMPK (p-AMPK),NF-κB p65,MCP-1,ICAM-1,and TGF-β1 protein.Results After stimulated by HG,the expression of NF-κB,MCP-1,ICAM-1,TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG (P <0.05).Both genes and protein expression of NF-κB,MCP-1,ICAM-1,TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner (P <0.05).The expression of p-AMPK increased with the rising of metformin concentration,presenting the opposite trend,while the level of total-AMPK protein was unchanged with exposure to HG or metformin.Conlusion Metformin can suppress the expression of NF-κB,MCP-1,ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation,which may partlv contribute to its reno-protection.

  6. Reduced fructosamine-3-kinase activity and its mRNA in human distal colorectal carcinoma.

    Science.gov (United States)

    Notarnicola, M; Caruso, Maria G; Tutino, V; Guerra, V; Frisullo, S; Altomare, D F; Misciagna, G

    2010-09-01

    Fructosamine-3-Kinase (FN3K) is an enzyme phosphorilating fructoselysine (FL) residues on glycated proteins, resulting in the production of protein-bound FL-3-phosphate. The pathological role of the non-enzymatic modification of proteins by reducing sugars has become increasingly evident in various types of disorders, including the cancer. In this study, our aim was to study FN3K enzyme activity, as well as its mRNA in human colorectal cancer (CRC). Thirty consecutive CRC patients undergoing surgery of the colon were enrolled in the study. FN3K enzymatic activity and gene expression were analyzed using a radiometric assay and quantitative RT-PCR, respectively. FN3K is a functionally active enzyme in human colon tissue, without significant differences between normal mucosa and cancer. The mean level of FN3K mRNA was significantly lower in cancer than in the corresponding normal colorectal mucosa The colorectal tumors located on the left side showed lower levels of both enzymatic activity and mRNA FN3K than tumors located in the right side of colon. This paper is the first studying FN3K enzyme activity in human CRC, showing a significant relationship between enzymatic activity, its mRNA and tumor side.

  7. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    Energy Technology Data Exchange (ETDEWEB)

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  8. Ablation of adenosine monophosphate-activated protein kinaseα1 in vascular smooth muscle cells promotes diet-induced atherosclerotic calcification in vivo

    Institute of Scientific and Technical Information of China (English)

    CAI Zhe-jun; DING Ye; ZHANG Miao; LU Qiu-lun; WU Sheng-nan; ZHU Huai-ping; SONG Ping; ZOU Ming-hui

    2016-01-01

    AIM:Atherosclerotic calcification is highly linked with plaque instability and cardiovascular events .Adenosine monophosphate-activated protein kinase ( AMPK) has been involved in the pathogenesis of various cardiovascular disease .The contributions of AMPKαsubunits to the development of atherosclerotic calcification in vivo remained unknown .We hypothesized that AMPKαsubunits may play a role in the development of atherosclerotic calcification .METHODS: Atherosclerotic calcification was generated by 24-week fed of western diet in ApoE-/-background mice .Calcification was evaluated in aortic roots and innominate arteries of ApoE-/-mice or in mice with dual deficiencies of ApoE and AMPKαsubunits globally ( AMPKα1 and AMPKα2 ) , or vascular smooth muscle cell ( VSMC)-specific or macrophage-specific knockout of AMPKα1 with atherosclerotic calcification pone diet . The mechanism of AMPKα1 in regulating Runx2 was further explored in human aortic VSMC .RESULTS: Ablation of AMPKα1 but not AMPKα2 in ApoE-/-background promoted atherosclerotic calcification with increased Runt -related transcription factor ( Runx2 ) expression in VSMC compared with ApoE-/-mice.Conversely, chronic administration of metformin, which activated AMPK, markedly reduced ath-erosclerotic calcification and Runx2 expression in ApoE-/-mice but had less effects in ApoE-/-/AMPKα1 -/-mice.Furthermore, VSMC-but not macrophage-specific deficiency of AMPKα1 in ApoE-/-background promoted atherosclerotic calcification in vivo com-pared with the controls .AMPKα1 silencing in human aortic VSMC prevented Runx 2 from proteasome degradation to trigger osteoblastic differentiation of VSMC .Conversely , activation of AMPK led to Runx 2 instability by inducing its small ubiquitin-like modifier modifi-cation (SUMOylation).Protein inhibitor of activated STAT-1 (PIAS1), the SUMO E3-ligase of Runx2, was directly phosphorylated by AMPKα1 at serine 510, to enhance its SUMO E3-ligase activity.Ablation of PIAS1

  9. Selective modulation of protein kinase isozymes by the site-selective analog 8-chloroadenosine 3',5'-cyclic monophosphate provides a biological means for control of human colon cancer cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Ally, S.; Tortora, G.; Clair, T.; Grieco, D.; Merlo, G.; Katsaros, D.; Ogreid, D.; Doeskeland, S.O.; Jahnsen, T.; Cho-Chung, Yoonsang

    1988-09-01

    Differential expression of type I and type II cAMP-dependent protein kinase isozymes has been linked to growth regulation and differentiation. The authors examined the expression of protein kinase isozymes in the LS 174T human colon cancer cell line during 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP)-induced growth inhibition. Two species of R/sup II/ (the regulatory subunit of protein kinase type II) with apparent M/sub r/ 52,000 (R/sup II//sub 52/) and M/sub r/ 56,000 (R/sup II//sub 56/) and a single species of R/sup I/ (the regulatory subunit of protein kinase type I) with M/sub r/ 48,000 were identified in the cancer cells. R/sup I/ and both forms of R/sup II/ were covalently labeled with 8-azidoadenosine 3',5'-cyclic (/sup 32/P)monophosphate, and two anti-R/sup II/ antibodies that exclusively recognize either R/sup II//sub 52/ or R/sup II//sub 56/ resolved two forms of the R/sup II/ receptors. 8-Cl-cAMP caused transcriptional activation of the R/sup II//sub 52/ receptor gene and inactivation of the R/sup I/ receptor gene. Thus, differential regulation of various forms of cAMP receptor proteins is involved in 8-Cl-cAMP-induced regulation of cancer cell growth, and nuclear translocation of R/sup II//sub 52/ receptor protein appears to be an early event in such differential regulation.

  10. Oncogenic kinase NPM/ALK induces expression of HIF1α mRNA.

    Science.gov (United States)

    Marzec, M; Liu, X; Wong, W; Yang, Y; Pasha, T; Kantekure, K; Zhang, P; Woetmann, A; Cheng, M; Odum, N; Wasik, M A

    2011-03-17

    The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1α (HIF1α) mRNA, even under normoxic conditions, and markedly upregulate HIF1α protein expression under hypoxia. HIF1α expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1α expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1α gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1α gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1α increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1α, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

  11. Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

    Science.gov (United States)

    Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

    2017-07-01

    Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

  12. Irisin ameliorates hepatic glucose/lipid metabolism and enhances cell survival in insulin-resistant human HepG2 cells through adenosine monophosphate-activated protein kinase signaling.

    Science.gov (United States)

    So, Wing Yan; Leung, Po Sing

    2016-09-01

    Irisin is a newly identified myokine that promotes the browning of white adipose tissue, enhances glucose uptake in skeletal muscle and modulates hepatic metabolism. However, the signaling pathways involved in the effects on hepatic glucose and lipid metabolism have not been resolved. This study aimed to examine the role of irisin in the regulation of hepatic glucose/lipid metabolism and cell survival, and whether adenosine monophosphate-activated protein kinase (AMPK), a master metabolic regulator in the liver, is involved in irisin's actions. Human liver-derived HepG2 cells were cultured in normal glucose-normal insulin (NGNI) or high glucose-high insulin (HGHI/insulin-resistant) condition. Hepatic glucose and lipid metabolism was evaluated by glucose output and glycogen content or triglyceride accumulation assays, respectively. Our results showed that irisin stimulated phosphorylation of AMPK and acetyl-CoA-carboxylase (ACC) via liver kinase B1 (LKB1) rather than Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) in HepG2 cells. Irisin ameliorated hepatic insulin resistance induced by HGHI condition. Irisin reduced hepatic triglyceride content and glucose output, but increased glycogen content, with those effects reversed by dorsomorphin, an AMPK inhibitor. Furthermore, irisin also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and promoted cell survival in an AMPK-dependent manner. In conclusion, our data indicate that irisin ameliorates dysregulation of hepatic glucose/lipid metabolism and cell death in insulin-resistant states via AMPK activation. These findings reveal a novel irisin-mediated protective mechanism in hepatic metabolism which provides a scientific basis for irisin as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes mellitus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    Science.gov (United States)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  14. Antidepressant drug exposure is associated with mRNA levels of tyrosine receptor kinase B in major depressive disorder.

    Science.gov (United States)

    Bayer, T A; Schramm, M; Feldmann, N; Knable, M B; Falkai, P

    2000-08-01

    1. Recent studies have provided support for the notion that the high affinity neurotrophin receptor tyrosine receptor kinase B (trk B) may be involved in the treatment of depression. 2. Using a quantitative RT-PCR approach trk B mRNA levels were determined in brain material from cerebellum, temporal cortex, and frontal cortex of control specimen and patients with major depressive disorder, schizophrenia and bipolar disorder (15 subjects each). 3. Interestingly, elevated trk B mRNA levels were found in cerebellum (3.6-fold) in patients with major depressive disorder, reaching statistical significance (p=0.03). 4. The major depressive disorder-on drugs group differed from controls (p=0.006) in the cerebellum. 5. Since only patients with major depressive disorder received antidepressants, elevated trk B mRNA levels are possibly related to drug treatment.

  15. Tissue kallikrein reverses insulin resistance and attenuates nephropathy in diabetic rats by activation of phosphatidylinositol 3-kinase/protein kinase B and adenosine 5'-monophosphate-activated protein kinase signaling pathways.

    Science.gov (United States)

    Yuan, Gang; Deng, Juanjuan; Wang, Tao; Zhao, Chunxia; Xu, Xizheng; Wang, Peihua; Voltz, James W; Edin, Matthew L; Xiao, Xiao; Chao, Lee; Chao, Julie; Zhang, Xin A; Zeldin, Darryl C; Wang, Dao Wen

    2007-05-01

    We previously reported that iv delivery of the human tissue kallikrein (HK) gene reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with insulin resistance. In the current study, we evaluated the potential of a recombinant adeno-associated viral vector expressing the HK cDNA (rAAV-HK) as a sole, long-term therapy to correct insulin resistance and prevent renal damage in streptozotocin-induced type-2 diabetic rats. Administration of streptozotocin in conjunction with a high-fat diet induced systemic hypertension, diabetes, and renal damage in rats. Delivery of rAAV-HK resulted in a long-term reduction in blood pressure, and fasting plasma insulin was significantly lower in the rAAV-HK group than in the control group. The expression of phosphatidylinositol 3-kinase p110 catalytic subunit and the levels of phosphorylation at residue Thr-308 of Akt, insulin receptor B, and AMP-activated protein kinases were significantly decreased in organs from diabetic animals. These changes were significantly attenuated after rAAV-mediated HK gene therapy. Moreover, rAAV-HK significantly decreased urinary microalbumin excretion, improved creatinine clearance, and increased urinary osmolarity. HK gene therapy also attenuated diabetic renal damage as assessed by histology. Together, these findings demonstrate that rAAV-HK delivery can efficiently attenuate hypertension, insulin resistance, and diabetic nephropathy in streptozotocin-induced diabetic rats.

  16. Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

    Science.gov (United States)

    Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

    2015-12-15

    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy.

  17. Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

    Science.gov (United States)

    Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan

    2015-03-25

    This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

  18. Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

    Science.gov (United States)

    Taibi, N; Dupont, J; Bouguermouh, Z; Froment, P; Ramé, C; Anane, A; Amirat, Z; Khammar, F

    2017-03-01

    In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent.

  19. MiR-506 suppresses liver cancer angiogenesis through targeting sphingosine kinase 1 (SPHK1) mRNA.

    Science.gov (United States)

    Lu, Zhanping; Zhang, Weiying; Gao, Shan; Jiang, Qiulei; Xiao, Zelin; Ye, Lihong; Zhang, Xiaodong

    MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3'-untranslated region (3'UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3'UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis.

  20. Steady-state concentrations of mRNA encoding two inhibitors of protein kinase C in ovine luteal tissue.

    Science.gov (United States)

    Juengel, J L; Melner, M H; Clapper, J A; Turzillo, A M; Moss, G E; Nett, T M; Niswender, G D

    1998-07-01

    Prostaglandin F2 alpha (PGF2 alpha) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF2 alpha to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF2 alpha might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF2 alpha receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF2 alpha receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF2 alpha, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF2 alpha receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml-1) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF2 alpha during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF2 alpha during maternal recognition of pregnancy

  1. Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

    Science.gov (United States)

    Cameron, Kimberly O; Kung, Daniel W; Kalgutkar, Amit S; Kurumbail, Ravi G; Miller, Russell; Salatto, Christopher T; Ward, Jessica; Withka, Jane M; Bhattacharya, Samit K; Boehm, Markus; Borzilleri, Kris A; Brown, Janice A; Calabrese, Matthew; Caspers, Nicole L; Cokorinos, Emily; Conn, Edward L; Dowling, Matthew S; Edmonds, David J; Eng, Heather; Fernando, Dilinie P; Frisbie, Richard; Hepworth, David; Landro, James; Mao, Yuxia; Rajamohan, Francis; Reyes, Allan R; Rose, Colin R; Ryder, Tim; Shavnya, Andre; Smith, Aaron C; Tu, Meihua; Wolford, Angela C; Xiao, Jun

    2016-09-08

    Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

  2. Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

    Science.gov (United States)

    Chen, Haiyan; Wang, Ji-Ping; Santen, Richard J; Yue, Wei

    2015-06-01

    Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

  3. Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA.

    Science.gov (United States)

    Perrone-Bizzozero, N I; Cansino, V V; Kohn, D T

    1993-03-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.

  4. Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

    Science.gov (United States)

    Wilson, Gabriel J; Moulton, Christopher J; Garlick, Peter J; Anthony, Tracy G; Layman, Donald K

    2012-11-13

    Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced

  5. Post-Meal Responses of Elongation Factor 2 (eEF2 and Adenosine Monophosphate-Activated Protein Kinase (AMPK to Leucine and Carbohydrate Supplements for Regulating Protein Synthesis Duration and Energy Homeostasis in Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Donald K. Layman

    2012-11-01

    Full Text Available Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2. This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0 or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270, 80:40:40 mg leucine, isoleucine, and valine (Leu80, 2.63 g carbohydrates (CHO2.6, 1 g carbohydrates (CHO1.0, or water (Sham control. Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK, acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0, but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to

  6. Adenosine monophosphate-activated protein kinase and myofibrillar protein degradation%腺苷酸活化蛋白激酶与骨骼肌蛋白质降解

    Institute of Scientific and Technical Information of China (English)

    马延超; 朱荣; 李俊平

    2012-01-01

    BACKGROUND: Adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor in skeletal muscle, which can be activated by exercise. AMPK, widely existing in eucaryotic cells, is the serine/threonine protein kinase. OBJECTIVE: To review the structure and the function of AMPK, changes of AMPK activity and the influence of AMPK activity on skeletal muscle protein degeneration during exercise. METHODS: A computer-based online retrieval of China National Knowledge Infrastructure (CNKI), Vip database, http://highwire.stanford.edu and www.ncbi.nlm.nih.gov/pubmed was performed to search papers regarding AMPK and myofibrillar protein degradation. The structure and the function of AMPK, the change of AMPK activity in exercise, and the effect of AMPK activation on myofibrillar protein degradation were retrieved. RESULTS AND CONCLUSION: A total of 35 papers were retrieved. This study summarized the structure and the function of AMPK. In the resistance exercise and in the moderate and high intensity cycle exercise, AMPK activity may be increased, and in the low intensity cycle exercise, AMPK activity may not be increased. Activated AMPK may promote the protein degradation.%背景:机体运动时骨骼肌收缩,ATP被大量消耗,产生大量腺苷一磷酸,导致腺苷酸活化蛋白激酶的激活.目的:综述不同运动过程中腺苷酸活化蛋白激酶活性的变化,以及腺苷酸活化蛋白激酶对骨骼肌蛋白质降解的研究成果.方法:检索中国期刊网、维普期刊数据库、www.ncbi.nlm.nih.gov/pubmed和http://highwire.stanford.edu/网站与腺苷酸活化蛋白激酶、运动、蛋白质降解研究相关的文章.并对腺苷酸活化蛋白激酶的结构与作用,不同运动过程中腺苷酸活化蛋白激酶活性的变化,以及腺苷酸活化蛋白激酶升高对骨骼肌蛋白质降解的内容进行分析综述.结果与结论:共纳入相关文献35篇.本文综述了腺苷酸活化蛋白激酶的结构、作用

  7. The upstream open reading frame of cyclin-dependent kinase inhibitor 1A mRNA negatively regulates translation of the downstream main open reading frame

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung Mi; Cho, Hana [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.

  8. Aurora kinase A is not involved in CPEB1 phosphorylation and cyclin B1 mRNA polyadenylation during meiotic maturation of porcine oocytes.

    Science.gov (United States)

    Komrskova, Pavla; Susor, Andrej; Malik, Radek; Prochazkova, Barbora; Liskova, Lucie; Supolikova, Jaroslava; Hladky, Stepan; Kubelka, Michal

    2014-01-01

    Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

  9. Aurora kinase A is not involved in CPEB1 phosphorylation and cyclin B1 mRNA polyadenylation during meiotic maturation of porcine oocytes.

    Directory of Open Access Journals (Sweden)

    Pavla Komrskova

    Full Text Available Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1. The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

  10. Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities.

    Science.gov (United States)

    Rapala-Kozik, Maria; Olczak, Mariusz; Ostrowska, Katarzyna; Starosta, Agata; Kozik, Andrzej

    2007-12-01

    A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis.

  11. 辛伐他汀抑制肝星状细胞活化及其对腺苷酸活化蛋白激酶活性的影响%Simvastatin inhibits activation of hepatic stellate cells and promotes activation of adenosine monophosphate activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    曹伟; 闫蕾; 王玮; 赵彩彦

    2012-01-01

    the underlying molecular mechanism ofthe cholesterol-blocking drug,simvastatin,in treating nonalcoholic fatty liver fibrosis.Method A rat model of nonalcoholic fatty liver fibrosis was established by feeding Wistar rats a fat-rich diet.After treatment with simvastatin (4 mg/kg/day),liver histological specimens were stained with hematoxylin-eosin and Masson's trichrome for microscopic analysis.Expression of adenosine monophosphate-activated protein kinase-alpha (AMPKα) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR; for mRNA) and Western blotting (protein).The levels of serum total cholesterol (TC),triglycerides (TG),alanine aminotransferase (ALT),aspartate aminotransferase (AST),and tumor necrosis factor-alpha (TNFa) were measured by standard biochemical assays.The human hepatic stellate cell line,LX-2 (quiescent or activated),was treated with transforming growth factor-beta l (TGF-β1) alone,simvastatin alone,or TGF-β1 +simvastatin.RT-PCR and Western blotting were used to determine changes in AMPKα mRNA and protein expression,respectively.Results In the rat model of nonalcoholic fatty liver fibrosis,the extent of pathological changes in hepatic tissues correlated with severity of disease progression.The levels of serum TC,TG,ALT,AST and TNFα were increased significantly in model rats (vs.healthy controls; all,P< 0.01).AMPKα mRNA expression and activity was significantly decreased in model rats (vs.healthy controls; P< 0.01 and P< 0.05,respectively).Simvastatin,treatment significantly improved all of these parameters in model rats (vs.untreated model rats; all,P< 0.05).In vitro simvastatin treatment of human HSCs significantly increased AMPKα activity (quiescent LX-2:0.93 -0.10 vs.0.72±0.09,activated LX-2:0.72±0.10 vs.0.54±0.10,q=7.00,6.00; all,P<0.01),decreased a-smooth muscle actin expression (mRNA:0.30±0.02 vs.0.36±0.02,protein:0.30±0.03 vs.0.38±0.02,q=11.245,11.216; all,P<0.01),and decreased collagen I expression

  12. An acute increase in fructose concentration increases hepatic glucose-6-phosphatase mRNA via mechanisms that are independent of glycogen synthase kinase-3 in rats.

    Science.gov (United States)

    Wei, Yuren; Bizeau, Michael E; Pagliassotti, Michael J

    2004-03-01

    It appears that low amounts of fructose improve, whereas increased concentrations impair glucose tolerance and hepatic glucose metabolism. In this study, we compared directly the effects of low vs. high portal vein fructose concentrations on hepatic glucose metabolism in rats, using glucose-6-phosphatase gene expression as an endpoint. In the control group (C; n = 7), pancreatic clamps were performed using somatostatin and replacement of insulin such that basal glucose levels were maintained. In the experimental groups (n = 8/group), hyperglycemic, hyperinsulinemic pancreatic clamps were performed in which glucose (G) or glucose + fructose was infused into a jejunal vein. Fructose was infused to achieve either low (F1; 1.0 mmol/L) portal vein concentrations. Total sugar load to the liver was equalized among the 3 experimental groups. Compared with C, liver glucose-6-phosphatase catalytic subunit mRNA was reduced by approximately 55% in G and F1, whereas it was increased approximately 180% in F2. F2 did not differentially affect glucose-6-phosphate translocase or phosphoenolpyruvate carboxykinase mRNA levels in liver, nor kidney glucose-6-phosphatase catalytic subunit mRNA. Livers from the F2 group were characterized by an accumulation of pentose phosphate intermediates and reduced phosphorylation of glycogen synthase kinase-3 (active form). However, in separate studies (n = 5/group), the infusion of a glycogen synthase kinase-3 inhibitor did not prevent the effects of F2 on glucose-6-phosphatase gene expression. We hypothesize that elevated fructose concentrations, similar to levels achieved after ingestion of sucrose- or fructose-enriched meals, initiate signals within the liver that elicit selective changes in hepatic gene expression.

  13. Molecular cloning and in silico analysis of three somatic embryogenesis receptor kinase mRNA from date palm

    Directory of Open Access Journals (Sweden)

    Rekik Imen

    2013-01-01

    Full Text Available We report here the isolation and characterizations of three somatic embryogenesis receptor kinase (PhSERK genes from palm date by a rapid amplification of cDNA ends (RACE approach. PhSERKs belong to a small family of receptor kinase genes, share a conserved structure and extensive sequence homology with previously reported plant SERK genes. Sequence analysis of these genes revealed the sequence size of 11051 pb (PhSERK1, 7981 pb (PhSERK2 and 10510 pb (PhSERK3. The open reading frames of PhSERK1, PhSERK2 and PhSERK3 are 1914 pb, 1797 pb and 1719 pb respectively. PhSERKs belongs to the LRR-type cell surface RLKs, which possess a number of characteristic domains. These include an extracellular domain (EX containing a variable number of LRR units, signal pepetide (SP immediately followed by a single transmembrane domain (TM and an intracellular kinase domain. The phylogenetic tree shows that the protein PhSERK1, PhSERK2 and PhSERK3 clustered within monocots SERKs proteins groups. We also predicted the secondary and tertiary with ligand binding sites structure of the protein PhSERKs.

  14. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  15. Effects of ginsenoside on brain-derived neurotrophic factor and tyrosine kinase B mRNA expression in the hippocampal formation of aged rats

    Institute of Scientific and Technical Information of China (English)

    Hong Lai; Wensu Liu; Zhaosheng Li; Haihua Zhao; Yongli Lü

    2008-01-01

    BACKGROUND:There are a limited number of studies involving the effects of ginsenosides,the active component of ginseng,on expression of hippocampal TrkB mRNA in aged rats.OBJECTIVE:To observe expression of brain-derived neurotrophic factor(BDNF) and tyrosine kinase B (TrkB)mRNA in the hippocampal formation of aged rats,as well as changes after ginsenoside administrated.DESIGN,TIME AND SETTING:A randomized,controlled experiment was performed at the Department of Anatomy,College of Basic Medical Sciences,China Medical University in March 2005.MATERIALS:A total of 39 female,Wistar rats were randomly divided into 3 groups (n=13 each):young (3-5 months old),aged(27 months old),and ginsenoside group(received 25mg/kg/d ginsenoside in the drinking water between 17 and 27 months of age).METHODS:Following anesthesia,the rats were exsanguinated and perfused transcardially with chilled,heparinized,0.9% saline.The brains were removed and post-fixed in 40 g/L paraformaldehyde/phosphate buffer for 20 minutes,and further incubated in 30% sucrose/phosphate buffer overnight.MAIN OUTCOME MEASURES:In situ hybridization,immunohistochemistry,and image analysis were used to investigate expression of BDNF and Trk(B mRNA in the hippocampal formation.RESULTS:The expression levels of BDNF in the hippocampal CA3 and CA1 of aged rats was significantly less than the young group(t=2.879,1.814,1.984,P<0.05).BDNF expression was significantly greater in the dentate gyrus of the ginsenoside group,compared with the aging group(t=1.943,P<0.01).The expression of TrkB mRNA in the hippocampal CA3,CA1,and dentate gyrus of aged rats was less than the young group(t=3.540,3.629,17.905,P<0.01).TrkB mRNA expression in the CA3 region and dentate gyrus of the ginsenoside group was significantly greater compared with the aging group(t=1.293,3.386,P<0.05.0.01).CONCLUSION:BDNF and TrkB mRNA expression in the hippocampal formation were reduced in the aged group.However,ginsenosides can increase BDNF and TrkB mRNA

  16. Mixed lineage kinase 3 inhibits platelet-derived growth factor-stimulated DNA synthesis and matrix mRNA expression in mesangial cells.

    Science.gov (United States)

    Parameswaran, Narayanan; Hall, Carolyn S; Böck, Barbara C; Sparks, Harvey V; Gallo, Kathleen A; Spielman, William S

    2002-01-01

    Mixed lineage kinase 3 (MLK 3) is a recently described member of the MLK subfamily of Ser/Thr protein kinases that interacts with MAPK pathways. The aim of this study was to test the potential interaction of MLK 3 with signaling pathways stimulated by PDGF in rat mesangial cells. We have established a stable cell line expressing human MLK 3 in rat glomerular mesangial cells. The effects of PDGF on proliferation and matrix mRNA expression were examined. In control (vector-transfected) mesangial cells PDGF increased [(3)H]-thymidine incorporation significantly in a concentration-dependent manner. In mesangial cells expressing MLK 3, PDGF-induced increase in DNA synthesis was significantly reduced. PDGF also induced fibronectin and collagen I mRNA expression in control cells, the effects of which were also significantly blocked in MLK 3-transfected cells. To understand the potential interaction of MLK 3 over expression with the MAPK pathways and to examine the potential mechanism of the effects of MLK 3 over expression on proliferation and matrix expression, activation of ERK2, JNK1 and p38 were examined. ERK2 activation was increased several fold by PDGF in control cells but was attenuated significantly in MLK 3 expressing cells. PDGF did not have any effect on JNK and p38 activation, in either cell types. Using the same stable-transfected cell line, identical results were obtained on proliferation and matrix expression with sarafotoxin-s6b (endothelin receptor agonist) another potent mitogenic and sclerotic agent for mesangial cells. These results indicate an important role for MLK 3 in the regulation of growth and matrix expression in mesangial cells.

  17. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    Science.gov (United States)

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G

    2014-03-05

    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

  18. A Cistanches Herba Fraction/β-Sitosterol Causes a Redox-Sensitive Induction of Mitochondrial Uncoupling and Activation of Adenosine Monophosphate-Dependent Protein Kinase/Peroxisome Proliferator-Activated Receptor γ Coactivator-1 in C2C12 Myotubes: A Possible Mechanism Underlying the Weight Reduction Effect

    Directory of Open Access Journals (Sweden)

    Hoi Shan Wong

    2015-01-01

    Full Text Available Previous studies have demonstrated that HCF1, a semipurified fraction of Cistanches Herba, causes weight reduction in normal diet- and high fat diet-fed mice. The weight reduction was associated with the induction of mitochondrial uncoupling and changes in metabolic enzyme activities in mouse skeletal muscle. To further investigate the biochemical mechanism underlying the HCF1-induced weight reduction, the effect of HCF1 and its active component, β-sitosterol (BSS, on C2C12 myotubes was examined. Incubation with HCF1/BSS caused a transient increase in mitochondrial membrane potential (MMP, possibly by fluidizing the mitochondrial inner membrane. The increase in MMP was paralleled to an increase in mitochondrial reactive oxygen species (ROS production. Mitochondrial ROS, in turn, triggered a redox-sensitive induction of mitochondrial uncoupling by uncoupling protein 3 (UCP3. Biochemical analysis indicated that HCF1 was capable of activating an adenosine monophosphate-dependent protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 pathway and thereby increased the expression of cytochrome c oxidase and UCP3. Animal studies using mitochondrial recoupler also confirmed the role of mitochondrial uncoupling in the HCF1-induced weight reduction. In conclusion, a HCF1/BSS causes the redox-sensitive induction of mitochondrial uncoupling and activation of AMPK/PGC-1 in C2C12 myotubes, with resultant reductions in body weight and adiposity by increased energy consumption.

  19. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia.

    Science.gov (United States)

    Abraham, E; Arcaroli, J; Shenkar, R

    2001-01-01

    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

  20. Expression of a novel pyridoxal kinase mRNA splice variant, PKH-T, in human testis

    Institute of Scientific and Technical Information of China (English)

    XingFang; Zuo-MinZhou; LiLu; Lan-LanYin; Jian-MinLi; YinZhen; HuiWang; Jia-HaoSha

    2004-01-01

    Aim: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa.Methods: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template.The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results:A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene,HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different develop-mental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients.Conclusion: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.

  1. The beneficial effects of betaine on dysfunctional adipose tissue and N6-methyladenosine mRNA methylation requires the AMP-activated protein kinase α1 subunit.

    Science.gov (United States)

    Zhou, Xihong; Chen, Jingqing; Chen, Jin; Wu, Weiche; Wang, Xinxia; Wang, Yizhen

    2015-12-01

    The current study was conducted to determine whether betaine could improve fatty acid oxidation, mitochondrial function and N6-methyladenosine (m(6)A) mRNA methylation in adipose tissue in high-fat-induced mice and how AMP-activated protein kinase α1 subunit (AMPKα1) was involved. AMPKα1 knockout mice and wild-type mice were fed either a low-fat diet, high-fat diet or high-fat diet supplemented with betaine in the drinking water for 8weeks. Our results showed that mitochondrial genes (PGC1α) and β-oxidation-related genes (CPT1a) at protein level were increased in wild-type mice supplemented with betaine when compared with those in mice with high-fat diet. Betaine also decreased FTO expression and improved m(6)A methylation in adipose tissue of wild-type mice with high-fat diet. However, betaine failed to exert the abovementioned effects in AMPKα1 knockout mice. In adipocytes isolated from mice with high-fat diet, betaine treatment increased lipolysis and lipid oxidation. Moreover, betaine decreased FTO expression and increased m(6)A methylation. However, while AMPKα1 was knockdown, no remarkable changes in adipocytes were observed under betaine treatment. Our results indicated that betaine supplementation rectified mRNA hypomethylation and high FTO expression induced by high-fat diet, which may contribute to its beneficial effects on impaired adipose tissue function. Our results suggested that the AMPKα1 subunit is required for the beneficial effects of betaine on dysfunctional adipose tissue and m(6)A methylation. These results may provide the foundation for a mechanism that links m(6)A methylation status in RNA, AMPKα1 phosphorylation and dysfunctional adipose tissue induced by high-fat diet.

  2. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    Science.gov (United States)

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  3. Plasma concentrations of the cyclic nucleotides, adenosine 3',5'-monophosphate and guanosine 3'.5'-monophosphate, in healthy adults treated with theophylline

    DEFF Research Database (Denmark)

    Fenger, M; Eriksen, P B; Andersen, O;

    1982-01-01

    Plasma concentrations of cyclic adenosine monophosphate and cyclic guanosine monophosphate were measured in 10 health adults before, during and after periods of theophylline administration. Cyclic adenosine monophosphate concentrations did not change significantly, but cyclic guanosine monophosph...

  4. Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds.

    Science.gov (United States)

    Yu, Jaeju; Saiardi, Adolfo; Greenwood, John S; Bewley, J Derek

    2014-05-01

    During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity.

  5. Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin

    DEFF Research Database (Denmark)

    Siemer, S; Ornskov, D; Guerra, B

    1999-01-01

    Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs...... cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after...

  6. Rapid, sequential activation of mitogen-activated protein kinases and transcription factors precedes proinflammatory cytokine mRNA expression in spleens of mice exposed to the trichothecene vomitoxin.

    Science.gov (United States)

    Zhou, Hui-Ren; Islam, Zahidul; Pestka, James J

    2003-03-01

    Since proinflammatory cytokine mRNA expression is induced within lymphoid tissue in vivo by the trichothecene vomitoxin (VT) in a rapid (1-2 h) and transient (4-8 h) fashion, it was hypothesized that mitogen-activated protein kinases (MAPKs) and transcription factors associated upstream with gene transcription of these cytokines are activated prior to or within these time windows. To test this hypothesis, mice were first treated with a single oral dose of VT and then analyzed for MAPK phosphorylation in the spleen. As little as 1 mg/kg of VT induced JNK 1/2, ERK 1/2, and p38 phosphorylation with maximal effects being observed at 5 to 100 mg/kg of VT. VT transiently induced JNK and p38 phosphorylation over a 60-min time period with peak effects being observed at 15 and 30 min, respectively. In contrast, ERK remained phosphorylated from 15 to 120 min. Next, the binding of activating protein 1 (AP-1), CCAAT enhancer-binding protein (C/EBP), CRE-binding protein (CREB), and nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay (EMSA) using four different consensus transcriptional control motifs at 0, 0.5, 1.5, 4, and 8 h after oral exposure to 25 mg/kg of VT. AP-1 binding activity was differentially elevated from 0.5 h to 8 h, whereas C/EBP binding was elevated only at 0.5 h. CREB binding decreased slightly at 0.5 h but gradually increased, reaching a maximum at 4 h. NF-kappaB binding was increased only slightly at 4 and 8 h. The specificities of AP-1, C/EBP, CREB, and NF-kappaB for relevant DNA motifs were verified by competition assays, using an excess of unlabeled consensus and mutant oligonucleotides. Supershift EMSAs and Western blot analysis identified specific VT-inducible DNA binding proteins for AP-1 (cJun, phospho c-jun, JunB, and JunD), C/EBP (C/EBPbeta), CREB (CREB-1 and ATF-2), and NF-kappaB (p50 and cRel). Finally, when the effects of oral VT exposure on proinflammatory gene expression were assessed at 3, 6, and 9 h

  7. Quantification of thymidine kinase (TK1) mRNA in normal and leukemic cells and investigation of structure-function relatiosnhip of recombinant TK1enzyme

    DEFF Research Database (Denmark)

    Kristensen, Tina

    patients with chronic lymphatic leukemia (CLL). 2: Structure-function relationship of recombinant TKI. In the first part a sensitive method (competitive PCR) for quantification of TKI mRNA was established. The TKI mRNA level was quantified in quiescent lymphocytes from control donors (n = 6......) and in lymphocytes stimulated to growth by the mitogen phytohemagglutinin. The expression in normal cells was compared with the level of TK1 mRNA level in patients with chronic lymphatic leukemia (n = 5). The results for the six control donors show a very low level of TK1 mRNA (below 0.006~1 O6 copies mg-’ protein...

  8. Vimentin is a component of a complex that binds to the 5'-UTR of human heme-regulated eIF2α kinase mRNA and regulates its translation.

    Science.gov (United States)

    Chatterjee, Sangeeta; Panda, Amaresh C; Berwal, Sunil K; Sreejith, R K; Ritvika, Charu; Seshadri, Vasudevan; Pal, Jayanta K

    2013-03-01

    The human heme-regulated eIF2α kinase, also called the human heme-regulated inhibitor (hHRI) is significantly up-regulated particularly at the level of translation during stress. In this report we show that during lead-stress, the regulation of hHRI mRNA translation is mediated through its 5'-untranslated region (UTR) that interacts with specific trans-acting factors. Further, vimentin has been identified as one of the trans-acting factors that contribute to this regulation.

  9. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  10. Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

    Science.gov (United States)

    Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel

    2011-06-01

    The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

  11. AMPKα2基因克隆及其野生型和突变型真核表达载体的构建%Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors

    Institute of Scientific and Technical Information of China (English)

    罗招凡; 李芳萍; 丁鹤林; 程桦

    2009-01-01

    背景:实验表明,通过激活单磷酸腺苷激活的蛋白激酶(AMP-Activated Protein Kinase, AMPK)α2可以增加胰岛素的敏感性和骨骼肌葡萄糖的摄取,其有望成为预防和治疗2型糖尿病的新的生理和药理作用靶点.目的:克隆人的AMPKα2基因,并构建其野生型和突变型真核表达载体.设计:单一样本观察.时间及地点:实验于2007-04/2008-01在中山大学附属第二医院临床分子生物实验室完成.材料:QuikChange Ⅱ Site-Directed Mutagenesis Kit为Stratagene公司产品.真核表达载体pcDNA3.1(+),大肠杆菌DH5α为实验室保存.人骨胳肌组织来源于中山大学附属第二医院手术截肢患者,获患者知情同意,新鲜取材后液氮冷冻.方法:采用反转录一聚合酶链反应技术从人骨骼肌扩增AMPKα2基因,并将其克隆到T载体,通过测序对其进行鉴定.采用Quickchange定点诱变试剂盒对AMPKα2基因进行体外定点诱变,并将其野生和突变的编码基因亚克隆到真核表达载体pcDNA3.1中,通过酶切和测序进行鉴定.主要观察指标:①目的基因的克隆.②定点诱变.③真核表达质粒的构建.结果:成功克隆了AMPKα2基因,大小约1 700 bp,与已发表的AMPKα2同源性为99%,GenBank录入号EF056019.成功将AMPKα2第45位Lysine(AAA)突变为Arginine(AGA),成功构建了野生型和突变型pcDNA-AMPK α2重组质粒.结论:实验成功克隆了AMPKα2基因,构建了其野生型和突变型真核表达载体.%BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation

  12. Renoprotective Effects of Fenofibrate via Modulation of LKB1/AMPK mRNA Expression and Endothelial Dysfunction in a Rat Model of Diabetic Nephropathy.

    Science.gov (United States)

    Al-Rasheed, Nawal M; Al-Rasheed, Nouf M; Attia, Hala A; Al-Amin, Maha A; Al-Ajmi, Hanaa N; Hasan, Iman H; Mohamad, Raeesa A; Sinjilawi, Nasr A

    2015-01-01

    This study was conducted to investigate whether the renoprotective effects of fenofibrate are mediated via attenuation of endothelial dysfunction and modulating the mRNA expression of adenosine monophosphate-activated protein kinase (AMPK) and its downstream kinase liver kinase B1 (LKB1) in rats with diabetic nephropathy (DN). Diabetes was induced by a single intraperitoneal injection of streptozotocin (55 mg kg(-1)). Fenofibrate (100 mg kg(-1), p.o.) was given to diabetic rats daily for 12 weeks. Treatment with fenofibrate significantly improved the renal function as revealed by the significant reductions in urinary albumin excretion and serum levels of creatinine and urea, in addition to the significant increase in creatinine clearance compared with the diabetic control group. Hyperglycemia-induced oxidative damage was ameliorated by treatment with fenofibrate as indicated by the significantly increased levels of glutathione and catalase together with the significant decrease in lipid peroxidation. Administration of fenofibrate caused significant increases in renal nitric oxide (NO) production and mRNA expression of endothelial NO synthase (eNOS), AMPK and LKB1, reflecting improvement of endothelial function. Our results give further insights into the mechanisms underlying the protective role of fenofibrate in DN via modulation of AMPK, LKB1 and eNOS mRNA expression.

  13. Determination of mRNA, and protein levels of p53, MDM2 and protein kinase CK2 subunits in F9 cells after treatment with the apoptosis-inducing drugs cisplatin and carboplatin

    DEFF Research Database (Denmark)

    Siemer, S; Ornskov, D; Guerra, B

    1999-01-01

    Protein kinase CK2 is a pleiotropic serine/threonine kinase which has been shown to phosphorylate numerous substrates. Evidence is accumulating that CK2 may exist complexed to a variety of cellular proteins, e.g. p53, MDM2, and A-Raf. Here, we explored the effects of the chemotherapeutic drugs...... cisplatin and carboplatin on the mRNA and protein levels of p53, MDM2 and CK2 in a murine teratocarcinoma cell line F9. Northern and Western blot analyses were performed and the CK2 activity was determined. The degree of apoptosis after drug treatment was assessed using the TUNEL test. Six hours after...... cisplatin and carboplatin treatment, the RNA level of p53 dropped by 59% +/- 9% and 86% +/- 8% respectively, whereas the observed level of p53 protein rose to 7 and 10 times over the untreated control, respectively. Treatment with 33 microM cisplatin prompted apoptosis as early as 4 h after drug treatment...

  14. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    Yamauchi Mika

    2007-11-01

    Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

  15. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation.

    Science.gov (United States)

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J; Pogge von Strandmann, Lisa; Gritsenko, Marina A; Jacobs, Jon M; Moore, Patrick S; Chang, Yuan

    2016-07-26

    Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

  16. Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

    Science.gov (United States)

    Wang, Xiuying; Liu, Yulan; Wang, Shuhui; Pi, Dingan; Leng, Weibo; Zhu, Huiling; Zhang, Jing; Shi, Haifeng; Li, Shuang; Lin, Xi; Odle, Jack

    2016-10-01

    Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

  17. Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Parish Tanya

    2010-02-01

    Full Text Available Abstract Background Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM, lipomannan (LM and phosphatidylinosotol mannosides (PIMs in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. Results Mutants lacking either impA (Rv1604 or suhB (Rv2701c were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137 when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. Conclusions We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

  18. Coronary microembolization induced myocardial contractile dysfunction and tumor necrosis factor-α mRNA expression partly inhibited by SB203580 through a p38 mitogen-activated protein kinase pathway

    Institute of Scientific and Technical Information of China (English)

    LI Lang; QU Nan; LI Dong-hua; WEN Wei-ming; HUANG Wei-qiang

    2011-01-01

    Background The microemboli produced during spontaneous plaque rupture and ulceration and during coronary intervention will reduce coronary reserve and cause cardiac dysfunction. It is though that inflammation caused by the microinfarction induced by the microembolization may play an essential role. It is known that the activation of p38mitogen-activated protein kinases (MAPK) in both infected and non-infected inflammation in myocardium may cause a contractile dysfunction. But the relation between the activation of p38 MAPK and microembolization is still unknown.Methods Sprague-Dawley rats were randomly divided into three groups: Sham group, coronary microembolization (CME) group and SB203580 group (n=10 per group). CME rats were produced by injection of 42 μm microspheres into the left ventricle with occlusion of the ascending aorta. SB203580, a p38 MAPK inhibitor, was injected into the femoral vein after the injection of microspheres to make the SB203580 group. Left ventricular ejection fraction (LVEF) was determined by echocardiography. The protein concentration of P38 MAPK in the myocardium was assessed by Western blotting. The relative expression of mRNA for tumor necrosis factor (TNF)-a was assessed by the technique of semi-quantitative polymerase chain reaction amplification.Results LVEF was depressed at three hours up to 12 hours in the CME group. Increased p38 MAPK activity and TNF-α mRNA expression were observed in the CME group. The administration of SB203580 partly inhibited p38 MAPK activity,but did not fully depress the TNF-α expression, and partly preserved cardiac contractile function.Conclusions p38 MAPK is significantly activated by CME and the inhibition of p38 MAPK can partly depress the TNF-α expression and preserve cardiac contractile function.

  19. [Isolation of inosine-5'-monophosphate from fish muscles].

    Science.gov (United States)

    Tugaĭ, V A; Akulin, V N; Epshteĭn, L M

    1987-01-01

    Conditions for transformation of tissue adenosine-5'-monophosphate (AMP) into inosine-5'-monophosphate (IMP) with the aid of endogenic AMP-aminohydrolase are developed resting on the studied properties of AMP-aminohydrolase (EC 3.5.4.6) from saltwater fish muscles (one of the enzymes participating in the nucleotide metabolism). Sorption of the nucleotide is performed on the activated charcoals A gamma-3 A gamma-5 which eluate IMP from acid solutions. It reduces the process of isolation, permits application of the acid wash solutions to remove salts; the alkaline ethyl alcohol-aid elution at the subsequent stages accelerates the process of nucleotide concentration by means of vacuum evaporation. The suggested approaches allow developing a simple method of IMP production from fish tissues which diminishes the cost of preparation.

  20. Green tea catechins enhance norepinephrine-induced lipolysis via a protein kinase A-dependent pathway in adipocytes.

    Science.gov (United States)

    Chen, Shu; Osaki, Noriko; Shimotoyodome, Akira

    2015-05-22

    Green tea catechins have been shown to attenuate obesity in animals and humans. The catechins activate adenosine monophosphate-activated protein kinase (AMPK), and thereby increase fatty acid oxidation in liver and skeletal muscles. Green tea catechins have also been shown to reduce body fat in humans. However, the effect of the catechins on lipolysis in adipose tissue has not been fully understood. The aim of this study was to clarify the effect of green tea catechins on lipolysis in adipocytes and to elucidate the underlying mechanism. Differentiated mouse adipocyte cell line (3T3-L1) was stimulated with green tea catechins in the presence or absence of norepinephrine. Glycerol and free fatty acids in the media were measured. Phosphorylation of hormone-sensitive lipase (HSL) was determined by Western blotting, and the mRNA expression levels of HSL, adipose triglyceride lipase (ATGL), and perilipin were determined by quantitative RT-PCR. The cells were treated with inhibitors of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), or mitogen-activated protein kinase (MAPK) to determine the responsible pathway. Treatment of 3T3-L1 adipocytes with green tea catechins increased the level of glycerol and free fatty acids released into the media in the presence, but not absence, of norepinephrine, and increased the level of phosphorylated HSL in the cells. The catechins also increased mRNA and protein levels of HSL and ATGL. PKA inhibitor (H89) attenuated the catechin-induced increase in glycerol release and HSL phosphorylation. The results demonstrate that green tea catechins enhance lipolysis in the presence of norepinephrine via a PKA-dependent pathway in 3T3-L1 adipocytes, providing a potential mechanism by which green tea catechins could reduce body fat.

  1. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    Science.gov (United States)

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  2. Non-Viral Deoxyribonucleoside Kinases--Diversity and Practical Use

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  3. Thymidine kinase 1 regulatory fine-tuning through tetramer formation

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Clausen, Anders R.; Andersson, Karl-Magnus;

    2013-01-01

    Abstract: Thymidine kinase 1 (TK1) provides a crucial precursor, deoxythymidine monophosphate, for nucleic acid synthesis, and the activity of TK1 increases by up to 200-fold during the S-phase of cell division in humans. An important part of the regulatory checkpoints is the ATP and enzyme...

  4. Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.

    Science.gov (United States)

    Webster, C R; Anwer, M S

    1998-05-01

    Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid-induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5%+/-1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 micromol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2%+/-3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 micromol/L), glucagon (200 nmol/L), and a combination of forskolin (20 micromol/L) and 3-isobutyl-1-methylxanthine (20 micromol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP-induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 micromol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid-induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid-induced apoptosis appears to involve PKA, MAPK, and PI3K.

  5. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

    Science.gov (United States)

    McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.

    1995-01-01

    Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of

  6. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

    Science.gov (United States)

    McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.

    1995-01-01

    Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of

  7. 5'-腺苷磷酰硫酸激酶及其R68K突变体对5'-腺苷-磷酸3'羟基的磷酸化%Phosphorylation of 3'-hydroxyl Group of 5'-adenosine Monophosphate by Adenosine 5'-phosphosulfate Kinase and Its R68K Mutant

    Institute of Scientific and Technical Information of China (English)

    杨洋; 黄园波; 马建辉; 孙梅好

    2013-01-01

    Sulfur,an essential element for all organisms,plays its biological roles mainly with valence starus of-2 and +6.Intracellular sulfate activating,first step for the assimilation of sulfate,includes biosynthesis of adenosine 5'-phosphosulfate (APS)catalyzed by ATP sulfurylase (ATPS) with side product pyrophosphate (PPi) and the subsequent phosphorylation of APS by adenosine 5'-phosphosulfate kinase(APSK) to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS),the universal sulfuryl group donor.Although the mechanism of APS phosphorylation by APSK had been reported in detail,it remains to be elucidated that if the APS analogue-AMP could be phosphorylated by APSK.Preliminary studies showed that AMP could be phosphorylated by APSK with product 3'-phosphcadenosine 5'-phosphate (PAP).Structural analysis indicated that R68 would form hydrogen bonds with oxygen on phosphate and sulfate to stabilize the binding of APS.Shortening R group by R68K mutation would weaken the interaction to sulfate,strengthen interaction to phosphoate,and possibly discriminate APS from AMP.Our results clearly demonstrated that optimized substrate for APSK turned to AMP from APS,with 5 times enhancement of catalytic efficiency and binding affinity.R68K was also further successfully used as coupling enzymes to keep PAP concentration constant and eliminate product AMP in solution to facilitate the initial rate measurements for PAP hydrolysis activity catalyzed by yeast 3',5'-bisphosphate nucleotidase (YND).R68K was proved to be an alternating coupling enzyme to facilitate characterization of AMP producing enzymes.%硫作为生命活动的必需元素,主要以-2价和+6价发挥生物学功能.硫的同化代谢包括胞内活化、转移以及还原等反应.其活化是同化代谢的关键反应,包括ATP硫酸化酶(ATP sulfurylase,ATPS)催化硫酸盐与ATP反应生成腺苷-5'-磷酰硫酸(adenosine 5'-phosphosulfate,APS)和焦磷酸(pyrophosphate,PPi)以及腺苷-5'-磷酰硫酸激酶(adenosine 5

  8. Specific Interactions of Antitumor Metallocenes with Deoxydinucleoside Monophosphates

    Science.gov (United States)

    Eberle, Rahel P.; Hari, Yvonne; Schürch, Stefan

    2017-09-01

    Bent metallocenes Cp2MCl2 (M = Ti, V, Nb, Mo) are known to exhibit cytotoxic activity against a variety of cancer types. Though the mechanism of action is not fully understood yet, the accumulation of the metal ions in the nucleus points towards DNA as one of the primary targets. A set of eight deoxydinucleoside monophosphates was used to study the adduct yields with metallocenes and cisplatin. The binding affinities are reflected by the relative intensities of the adducts and were found to follow the order of Pt > V > Ti > Mo (no adducts were detected with Nb). High-resolution tandem mass spectrometry was applied to locate the binding patterns in the deoxydinucleoside monophosphates. Whereas cisplatin binds to the soft nitrogen atoms in the purine nucleobases, the metallocenes additionally interact with the hard phosphate oxygen, which is in good agreement with the hard and soft (Lewis) acids and bases (HSAB) concept. However, the binding specificities were found to be unique for each metallocene. The hard Lewis acids titanium and vanadium predominantly bind to the deprotonated phosphate oxygen, whereas molybdenum, an intermediate Lewis acid, preferentially interacts with the nucleobases. Nucleobases comprise alternative binding sites for titanium and vanadium, presumably oxygen atoms for the first and nitrogen atoms for the latter. In summary, the intrinsic binding behavior of the different metallodrugs is reflected by the gas-phase dissociation of the adducts. Consequently, MS/MS can provide insights into therapeutically relevant interactions between metallodrugs and their cellular targets. [Figure not available: see fulltext.

  9. Gemcitabine-based therapy for pancreatic cancer using the squalenoyl nucleoside monophosphate nanoassemblies.

    Science.gov (United States)

    Maksimenko, Andrei; Caron, Joachim; Mougin, Julie; Desmaële, Didier; Couvreur, Patrick

    2015-03-30

    Gemcitabine is currently the most effective agent against advanced pancreatic cancer. However, the major therapeutic hurdles using gemcitabine include rapid inactivation by blood deaminases and fast development of cell chemoresistance, induced by down-regulation of deoxycytidine kinase or nucleoside transporters. To overcome the above drawbacks we designed recently a novel nanomedicine strategy based on squalenoyl prodrug of 5'-monophosphate gemcitabine (SQdFdC-MP). This amphiphilic conjugate self-organized in water into unilamellar vesicles with a mean diameter of 100 nm. In this study the antitumor efficacy of SQdFdC-MP nanoassemblies (NAs) on chemoresistant and chemosensitive pancreatic adenocarcinoma models have been investigated. Cell viability assays showed that SQdFdC-MP NAs displayed higher antiproliferative and cytotoxic effects, particularly in chemoresistant pancreatic tumor cells. In in vivo studies, SQdFdC-MP NAs decreased significantly the growth (∼70%) of human MiaPaCa2 xenografts, also preventing tumor cell invasion, whereas native dFdC did not display any anticancer activity when tumor growth inhibition was only 35% with SQdFdC NAs. These results correlated with a reduction of Ki-67 antigen and the induction of apoptosis mediated by caspase-3 activation in tumor cells. These findings demonstrated the feasibility of utilizing SQdFdC-MP NAs to make tumor cells more sensitive to gemcitabine and thus providing an efficient new therapeutic alternative for pancreatic adenocarcinoma.

  10. Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling.

    Science.gov (United States)

    Sato, Masayuki J; Kuwayama, Hidekazu; van Egmond, Wouter N; Takayama, Airi L K; Takagi, Hiroaki; van Haastert, Peter J M; Yanagida, Toshio; Ueda, Masahiro

    2009-04-21

    Switching between attractive and repulsive migration in cell movement in response to extracellular guidance cues has been found in various cell types and is an important cellular function for translocation during cellular and developmental processes. Here we show that the preferential direction of migration during electrotaxis in Dictyostelium cells can be reversed by genetically modulating both guanylyl cyclases (GCases) and the cyclic guanosine monophosphate (cGMP)-binding protein C (GbpC) in combination with the inhibition of phosphatidylinositol-3-OH kinases (PI3Ks). The PI3K-dependent pathway is involved in cathode-directed migration under a direct-current electric field. The catalytic domains of soluble GCase (sGC) and GbpC also mediate cathode-directed signaling via cGMP, whereas the N-terminal domain of sGC mediates anode-directed signaling in conjunction with both the inhibition of PI3Ks and cGMP production. These observations provide an identification of the genes required for directional switching in electrotaxis and suggest that a parallel processing of electric signals, in which multiple-signaling pathways act to bias cell movement toward the cathode or anode, is used to determine the direction of migration.

  11. Turning an antiviral into an anticancer drug: nanoparticle delivery of acyclovir monophosphate.

    Science.gov (United States)

    Yao, Jing; Zhang, Yuan; Ramishetti, Srinivas; Wang, Yuhua; Huang, Leaf

    2013-09-28

    Anti-herpes simplex virus (HSV) drug acyclovir (ACV) is phosphorylated by the viral thymidine kinase (TK), but not the cellular TK. Phosphorylated ACV inhibits cellular DNA synthesis and kills the infected cells. We hypothesize that ACV monophosphate (ACVP), which is an activated metabolite of ACV, should be efficient in killing cells independent of HSV-TK. If so, ACVP should be a cytotoxic agent if properly delivered to the cancer cells. The Lipid/Calcium/Phosphate (LCP) nanoparticles (NPs) with a membrane/core structure were used to encapsulate ACVP to facilitate the targeted delivery of ACVP to the tumor. The LCP NPs showed entrapment efficiency of ~70%, the nano-scaled particle size and positive zeta potential. Moreover, ACVP-loaded LCP NPs (A-LCP NPs) exhibited concentration-dependent cytotoxicity against H460 cells and increased S-phase arrest. More importantly, a significant reduction of the tumor volume over 4 days following administration (pACV and ACVP) and blank LCP NPs showed little or no therapeutic effect. It was also found that the high efficacy of A-LCP NPs was associated with the ability to induce dramatic apoptosis of the tumor cells, as well as significantly inhibit tumor cell proliferation and cell cycle progression. In conclusion, with the help of LCP NPs, monophosphorylation modification of ACV can successfully modify an HSV-TK-dependent antiviral drug into an anti-tumor drug.

  12. The role of renal adenosine 3',5'-monophosphate in the control of erythropoietin production.

    Science.gov (United States)

    Rodgers, G M; Fisher, J W; George, W J

    1975-01-01

    A regulatory role for adenosine 3',5'-monophosphate (cyclic AMP) in the production of the renal hormone rythropoietin following erythropoietic stimulation with cobaltous chloride hexahydrate is proposed. Studies in rates reveal a temporal relationship between renal cyclic AMP levels and plasma titers of erythropoietin. In addition, cobalt increases the activity of an erythropoietin-generating enzyme (renal erythropoietic factor) with maximal enzyme activity occurring after the rise in cyclic AMP levels but before the increase in erythropoietin titers. This increase in renal cyclic AMP is localized to the renal cortex. Cobalt stimulates renal cortical adenylate cyclase but has no effect on renal cyclic nucleotide phosphodiesterase. The addition of cyclic AMP (3 time 10-6 M) and a partially purified cyclic AMP-dependent protein kinase from rat kidney to an inactive preparation of renal erythropoietic factor increases the ability of renal erythropoietic factor to generate erythropoietin. Data from the polycythemic mouse assay, a bioassay used to quantitate erythropoietic activity of test substances, indicate that dibutyryl cyclic AMP is erythropoietically active with respect to its ability to increase radioactive-labelled iron (59Fe) incorporation into heme of newly formed red blood cells. Theophylline, which by itself is erythropoietically inactive, potentiated the erythropoietic effect of cobalt in polycythemic mice. These results suggest that cyclic AMP plays a significant role in the renal production of erythropoietin following cobalt administration. It is postulated that cobalt stimulates renal cortical adenyoate cyclase, thus increasing renal cyclic AMP levels. Cyclic AMP then activates a protein kinase which subsequently stimulates renal erythropoietic factor to generate erythropoietin. A similar cyclic AMP mechanism may be operative after erythropoietic stimulation by exposure to hypoxia or prostaglandin treatment.

  13. Effects of plant extract neferine on cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in rabbit corpus cavernosum in vitro

    Institute of Scientific and Technical Information of China (English)

    Jun Chen; Ji-Hong Liu; Tao Wang; Heng-Jun Xiao; Chun-Ping Yin; Jun Yang

    2008-01-01

    Aim: To further investigate the relaxation mechanism of neferine (Nef), a bis-benzylisoquinoline alkaloid extracted (isolated) from the green seed embryo of Nelumbo nucifera Gaertn in China, on rabbit corpus cavernosum tissue in vitro. Methods: The effects of Nef on the concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in isolated and incubated rabbit corpus cavernosum tissue were re-corded using125Ⅰ radioimmunoassay. Results: The basal concentration of cAMP in corpus cavernosum tissue was 5.67±0.97 pmol/mg. Nef increased the cAMP concentration in a dose-dependent manner (P 0.05). The accumulation of cAMP induced by prostaglandin E1(PGE1, a stimulator of cAMP production) was also augmented by Nef in a dose-dependent manner (P 0.05). Also,sodium nitroprusside (SNP, a stimulator of cGMP production)-induced cGMP production was not enhanced by Nef (P > 0.05). Conclusion: Nef, with its relaxation mechanism, can enhance the concentration of cAMP in rabbit corpus cavernosum tissue, probably by inhibiting phosphodiesterase activity.

  14. Active Fe-Containing Superoxide Dismutase and Abundant sodF mRNA in Nostoc commune (Cyanobacteria) after Years of Desiccation

    Science.gov (United States)

    Shirkey, Breanne; Kovarcik, Don Paul; Wright, Deborah J.; Wilmoth, Gabriel; Prickett, Todd F.; Helm, Richard F.; Gregory, Eugene M.; Potts, Malcolm

    2000-01-01

    Active Fe-superoxide dismutase (SodF) was the third most abundant soluble protein in cells of Nostoc commune CHEN/1986 after prolonged (13 years) storage in the desiccated state. Upon rehydration, Fe-containing superoxide disumutase (Fe-SOD) was released and the activity was distributed between rehydrating cells and the extracellular fluid. The 21-kDa Fe-SOD polypeptide was purified, the N terminus was sequenced, and the data were used to isolate sodF from the clonal isolate N. commune DRH1. sodF encodes an open reading frame of 200 codons and is expressed as a monocistronic transcript (of approximately 750 bases) from a region of the genome which includes genes involved in nucleic acid synthesis and repair, including dipyrimidine photolyase (phr) and cytidylate monophosphate kinase (panC). sodF mRNA was abundant and stable in cells after long-term desiccation. Upon rehydration of desiccated cells, there was a turnover of sodF mRNA within 15 min and then a rise in the mRNA pool to control levels (quantity of sodF mRNA in cells in late logarithmic phase of growth) over approximately 24 h. The extensive extracellular polysaccharide (glycan) of N. commune DRH1 generated superoxide radicals upon exposure to UV-A or -B irradiation, and these were scavenged by SOD. Despite demonstrated roles for the glycan in the desiccation tolerance of N. commune, it may in fact be a significant source of damaging free radicals in vivo. It is proposed that the high levels of SodF in N. commune, and release of the enzyme from dried cells upon rehydration, counter the effects of oxidative stress imposed by multiple cycles of desiccation and rehydration during UV-A or -B irradiation in situ. PMID:10613879

  15. Strong inhibition of TNF-alpha production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor

    NARCIS (Netherlands)

    Westra, J; Doornbos-van der Meer, B; de Boer, Peter; van Leeuwen, MA; van Rijswijk, Martin; Limburg, PC

    2004-01-01

    In inflammatory processes, the p38 mitogen-activated protein kinase ( MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) is a pivotal cytokine in rheumatoid arthritis and its production in macroph

  16. Adenosine Monophosphate-Based Detection of Bacterial Spores

    Science.gov (United States)

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

    2009-01-01

    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  17. Vascular relaxation and cyclic guanosine monophosphate in hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Otsuka, Y.; DiPiero, A.; Lockette, W.

    1986-03-01

    Isolated aortae from hypertensive rats have a decreased relaxation response to acetylcholine (Ach), A23187, and nitroprusside (SNP). Since cyclic guanosine monophosphate (cGMP) has been shown to increase in response to these vasodilators, the authors measured cGMP in response to these agents in isolated aortae from normotensive rats and DOCA, 1K1C, and coarctation induced hypertension. cGMP was measured by radioimmunoassay in vessels after exposure to phenylephrine followed by either Ach, A23187, or SNP. The aortae from the hypertensive rats had decreased basal levels of cGMP and attenuated increases in cGMP in response to Ach and A23187. Rises in cGMP in response to SNP were also attenuated in aortae from the hypertensive rats, even at concentrations which induced similar relaxation in normotensive and hypertensive blood vessels. The data suggest that changes in cGMP do not necessarily reflect changes in endothelium independent vascular relaxation in hypertension.

  18. 5’-Monophosphate-activated protein kinase (AMPK) improves autophagic activity in diabetes and diabetic complications

    Institute of Scientific and Technical Information of China (English)

    Fan Yao; Ming Zhang; Li Chen

    2016-01-01

    Diabetes mellitus(DM),an endocrine disorder,will be one of the leading causes of death world-wide in about two decades.Cellular injuries and disorders of energy metabolism are two key factors in the pathogenesis of diabetes,which also become the important causes for the process of diabetic complications.AMPK is a key enzyme in maintaining metabolic homeostasis and has been implicated in the activation of autophagy in distinct tissues.An increasing number of researchers have confirmed that autophagy is a potential factor to affect or induce diabetes and its complications nowadays,which could remove cytotoxic proteins and dysfunctional organelles.This review will summarize the regulation of autophagy and AMPK in diabetes and its complications,and explore how AMPK stimulates autophagy in different diabetic syndromes.A deeper understanding of the regulation and activity of AMPK in autophagy would enhance its development as a promising therapeutic target for diabetes treatment.

  19. Lower urinary tract symptoms/benign prostatic hypertrophy and vascular function: Role of the nitric oxide-phosphodiesterase type 5-cyclic guanosine 3',5'-monophosphate pathway.

    Science.gov (United States)

    Higashi, Yukihito

    2017-06-01

    It is well known that there is an association of lower urinary tract symptoms/benign prostatic hypertrophy with cardiovascular disease, suggesting that lower urinary tract symptoms/benign prostatic hypertrophy is a risk factor for cardiovascular events. Vascular function, including endothelial function and vascular smooth muscle function, is involved in the pathogenesis, maintenance and development of atherosclerosis, leading to cardiovascular events. Vascular dysfunction per se should also contribute to lower urinary tract symptoms/benign prostatic hypertrophy. Both lower urinary tract symptoms/benign prostatic hypertrophy and vascular dysfunction have cardiovascular risk factors, such as hypertension, dyslipidemia, diabetes mellitus, aging, obesity and smoking. Inactivation of the phosphodiesterase type 5-cyclic guanosine 3',5'-monophosphate-nitric oxide pathway causes lower urinary tract symptoms/benign prostatic hypertrophy through an enhancement of sympathetic nervous activity, endothelial dysfunction, increase in Rho-associated kinase activity and vasoconstriction, and decrease in blood flow of pelvic viscera. Both endogenous nitric oxide and exogenous nitric oxide act as vasodilators on vascular smooth muscle cells through an increase in the content of cyclic guanosine 3',5'-monophosphate, which is inactivated by phosphodiesterase type 5. In a clinical setting, phosphodiesterase type 5 inhibitors are widely used in patients with lower urinary tract symptoms/benign prostatic hypertrophy. Phosphodiesterase type 5 inhibitors might have beneficial effects on vascular function through not only inhibition of cyclic guanosine 3',5'-monophosphate degradation, but also increases in testosterone levels and nitric oxide bioavailability, increase in the number and improvement of the function of endothelial progenitor cells, and decrease in insulin resistance. In the present review, the relationships between lower urinary tract symptoms/benign prostatic hypertrophy, the

  20. Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion

    DEFF Research Database (Denmark)

    Morgan-Fisher, Marie; Couchman, John R; Yoneda, Atsuko

    2013-01-01

    migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition...... II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively......The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II...

  1. Structural basis for the catalytic mechanism of a proficient enzyme: Orotidine 5'-Monophosphate Decarboxylase

    DEFF Research Database (Denmark)

    Harris, Pernille Hanne; Poulsen, Jens-Christian Navarro; Jensen, Kaj Frank

    2000-01-01

    Orotidine 5‘-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5‘-monophosphate, the last step in the de novo synthesis of uridine 5‘-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction...... rate by a factor of 1017. This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 Å resolution structure of ODCase complexed with the inhibitor 1-(5‘-phospho-ß-d-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two a...

  2. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Anders Ranegaard Clausen, Anders Ranegaard; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized...... in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2...

  3. Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Ali, Ashfaq

    2012-01-01

    Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized...... in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines....... Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2...

  4. An antisense oligodeoxynucleotide targeted against the type II sub. beta. regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    Energy Technology Data Exchange (ETDEWEB)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S. (National Institutes of Health, Bethesda, MD (USA))

    1990-01-01

    The type II{sub {beta}} regulatory subunit of cAMP-dependent protein kinase (RII{sub {beta}}) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII{sub {beta}} antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII{sub {beta}} antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII{sub {beta}} protein. Exposure to RII{sub {beta}} sense, RI{sub {alpha}} and RII{sub {alpha}} antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII{sub {beta}} regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells.

  5. Hindbrain raphe stimulation boosts cyclic adenosine monophosphate and signaling proteins in the injured spinal cord.

    Science.gov (United States)

    Carballosa-Gonzalez, Melissa M; Vitores, Alberto; Hentall, Ian D

    2014-01-16

    Early recovery from incomplete spinal cord contusion is improved by prolonged stimulation of the hindbrain's serotonergic nucleus raphe magnus (NRM). Here we examine whether increases in cyclic adenosine monophosphate (cAMP), an intracellular signaling molecule with several known restorative actions on damaged neural tissue, could play a role. Subsequent changes in cAMP-dependent phosphorylation of protein kinase A (PKA) and PKA-dependent phosphorylation of the transcription factor "cAMP response element-binding protein" (CREB) are also analyzed. Rats with moderate weight-drop injury at segment T8 received 2h of NRM stimulation beginning three days after injury, followed immediately by separate extraction of cervical, thoracic and lumbar spinal cord for immunochemical assay. Controls lacked injury, stimulation or both. Injury reduced cAMP levels to under half of normal in all three spinal regions. NRM stimulation completely restored these levels, while producing no significant change in non-injured rats. Pretreatment with the 5-HT7 receptor antagonist pimozide (1 mg/kg, intraperitoneal) lowered cAMP in non-injured rats to injury amounts, which were unchanged by NRM stimulation. The phosphorylated fraction of PKA (pPKA) and CREB (pCREB) was reduced significantly in all three regions after SCI and restored by NRM stimulation, except for pCREB in lumbar segments. In conclusion, SCI produces spreading deficits in cAMP, pPKA and pCREB that are reversible by Gs protein-coupled 5-HT receptors responding to raphe-spinal activity, although these signaling molecules are not reactive to NRM stimulation in normal tissue. These findings can partly explain the benefits of NRM stimulation after SCI. © 2013 Published by Elsevier B.V.

  6. Cyclic adenosine monophosphate signal pathway in targeted therapy of lymphoma

    Institute of Scientific and Technical Information of China (English)

    DOU Ai-xia; WANG Xin

    2010-01-01

    Objective To review the role of cyclic adenosine monophosphate (cAMP) signal pathway in the pathogenesis oflymphoma and explore a potential lymphoma therapy targeted on this signaling pathway.Data sources The data cited in this review were mainly obtained from the articles listed in Medline and PubMed,published from January 1995 to June 2009. The search terms were "cAMP" and "lymphoma".Study selection Articles regarding the role of the cAMP pathway in apoptosis of lymphoma and associated cells and itspotential role in targeted therapy of lymphoma.Results In the transformation of lymphocytic malignancies, several signal pathways are involved. Among of them, thecAMP pathway has attracted increasing attention because of its apoptosis-inducing role in several lymphoma cells. cAMPpathway impairment is found to influence the prognosis of lymphoma. Targeted therapy to the cAMP pathway seems tobe a new direction for lymphoma treatment, aiming at restoring the cAMP function.Conclusions cAMP signal pathway has different effects on various lymphoma cells. cAMP analogues andphosphodiesterase 4B (PDE4B) inhibitors have potential clinical significance. However, many challenges remain inunderstanding the various roles of such agents.

  7. [Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver].

    Science.gov (United States)

    Kolos, I K; Makarchikov, A F

    2014-01-01

    In animals, thiamine monophosphate (TMP) is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levamisole in uncompetitive manner with K1 of 53 μM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecular-weight protein phosphotyrosine phosphatase.

  8. Crystal structure of human nicotinamide riboside kinase.

    Science.gov (United States)

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  9. Crystal Structure of Human Nicotinamide Riboside Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  10. ALLERGEN-INDUCED CHANGES IN ADENOSINE 5'-MONOPHOSPHATE BRONCHIAL RESPONSIVENESS - EFFECT OF NEDOCROMIL SODIUM

    NARCIS (Netherlands)

    AALBERS, R; KAUFMAN, HF; GROEN, H; KOETER, GH; DEMONCHY, JGR

    1992-01-01

    Bronchial hyperresponsiveness to adenosine 5'-monophosphate (AMP) was studied after allergen challenge in allergic asthmatic patients. Measurements were made with and without nedocromil sodium pretreatment. Nedocromil sodium inhibited both the early and late asthmatic reactions (P <.01). After aller

  11. ALLERGEN-INDUCED CHANGES IN ADENOSINE 5'-MONOPHOSPHATE BRONCHIAL RESPONSIVENESS - EFFECT OF NEDOCROMIL SODIUM

    NARCIS (Netherlands)

    AALBERS, R; KAUFMAN, HF; GROEN, H; KOETER, GH; DEMONCHY, JGR

    1992-01-01

    Bronchial hyperresponsiveness to adenosine 5'-monophosphate (AMP) was studied after allergen challenge in allergic asthmatic patients. Measurements were made with and without nedocromil sodium pretreatment. Nedocromil sodium inhibited both the early and late asthmatic reactions (P <.01). After

  12. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    thymidine and towards other deoxynucleosides and nucleoside analogs. One of the HuTK1-mutants gained increased activity with the analog azidothymidine and reduced thymidine activity, and two CeTK1 mutants phosphorylates deoxycytidine, which has not previously been observed for TK1-type kinases. They did...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site....

  13. Schistosoma mansoni c-AMP-dependent Protein Kinase (PKA): A Potential New Drug Target

    Science.gov (United States)

    2009-12-07

    chloroadenosine 3’,5’-monophosphate in breast cancer patients and xenograft bearing mice. Ann Oncol 7: 291-296. 129. Tortora G, Ciardiello F, Pepe S...cyclic-AMP-dependent protein kinases by using cyclic nucleotide analogs. Eur J Biochem 181: 19-31. 47. Yokozaki H, Tortora G, Pepe S, Maronde E...181: 19-31. 150 28. Ally S, Tortora G, Clair T, Grieco D, Merlo G, et al. (1988) Selective modulation of protein kinase isozymes by the site

  14. Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver

    Directory of Open Access Journals (Sweden)

    I. K. Kolas

    2014-12-01

    Full Text Available In animals, thiamine monophosphate (TMP is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levami­sole in uncompetitive manner with Ki of 53 μM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent­ Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecu­lar-weight protein phosphotyrosine phosphatase

  15. Metformin directly inhibits ghrelin secretion through AMP-activated protein kinase in rat primary gastric cells.

    Science.gov (United States)

    Gagnon, J; Sheppard, E; Anini, Y

    2013-03-01

    The antidiabetic drug Metformin causes weight loss in both diabetic and non-diabetic individuals. Metformin treatment is also associated with lower circulating levels of the orexigenic hormone ghrelin. To test whether Metformin directly affects ghrelin cells, rat primary stomach cells were treated with Metformin and the levels of ghrelin secretion, proghrelin gene expression and activation of adenosine monophosphate-activated protein kinase (AMPK) were examined. Metformin significantly reduced ghrelin secretion and proghrelin mRNA production and both these effects were blocked by co-incubation with the AMPK inhibitor compound C. Furthermore, the AMPK activator 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) significantly inhibited ghrelin secretion. Additionally, ghrelin cells were shown to express AMPK. Finally, Metformin treatment caused a significant increase in the level of phosphorylated (active) AMPK. Our results show that Metformin directly inhibits stomach ghrelin production and secretion through AMPK. This reduction in ghrelin secretion may be one of the key components in Metformin's mechanism of weight loss.

  16. Response of AMP-activated protein kinase and energy metabolism to acute nitrite exposure in the Nile tilapia Oreochromis niloticus.

    Science.gov (United States)

    Xu, Zhixin; Li, Erchao; Xu, Chang; Gan, Lei; Qin, Jian G; Chen, Liqiao

    2016-08-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a prevalent mammalian energy metabolism sensor, but little is known about its role as an energy sensor in fish experiencing stress. We aimed to study AMPK in Oreochromis niloticus on both the molecular and the physical level. We found that the cDNAs encoding the AMPKα1 and AMPKα2 variants of the O. niloticus catalytic α subunit were 1753bp and 2563 bp long and encoded 571 and 557 amino acids, respectively. Both the AMPKα1 and the AMPKα2 isoform possess structural features similar to mammalian AMPKα, including a phosphorylation site at Thr172 in the N-terminus, and exhibit high homology with other fish and vertebrate AMPKα sequences (81.3%-98.1%). mRNA encoding the AMPKα isoforms was widely expressed in various tissues with distinctive patterns. AMPKα1 and AMPKα2 were primarily expressed in the intestines and brain, respectively. Under acute nitrite challenge, the mRNA encoding the AMPKα isoforms, as well as AMPK activity, changed over time. Its recovery period in freshwater, combined with the fact that it is highly conserved, suggests that fish AMPK, like its mammalian orthologues, acts as an energy metabolism sensor. Furthermore, subsequent decreases in AMPK mRNA levels and activity suggested that its action was transient but efficient. Physically, glucose, lactic acid and TGs in plasma, as well as energy materials in the hepatopancreas and muscle, were significantly altered over time, indicating changes in energy metabolism during the experimental period. These data have enabled us to characterize energy utilization in O. niloticus and further illustrate the role of fish AMPK as an energy sensor. This study provides new insight into energy metabolism and sensing by AMPK in teleost and necessitates further study of the multiple physiologic roles of AMPK in fish.

  17. Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae.

    Science.gov (United States)

    Harigaya, Yuriko; Parker, Roy

    2012-07-17

    The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3' to 5' degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5' monophosphate ends on mRNAs in wild-type and dcp2 xrn1 yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low-level endonucleolytic cleavage were observed, suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5' monophosphates on some mRNAs, suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested, Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential step to regulate gene expression.

  18. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen

    2017-05-31

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  19. Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.

    Science.gov (United States)

    Takeuchi, Masaki; Nagaoka, Yasutaka; Yamada, Toshimichi; Takakura, Hideo; Ozawa, Takeaki

    2010-11-15

    Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.

  20. Mutational analysis of UMP kinase from Escherichia coli.

    Science.gov (United States)

    Bucurenci, N; Serina, L; Zaharia, C; Landais, S; Danchin, A; Bârzu, O

    1998-02-01

    UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

  1. Squalenoyl nucleoside monophosphate nanoassemblies: new prodrug strategy for the delivery of nucleotide analogues.

    Science.gov (United States)

    Caron, Joachim; Reddy, L Harivardhan; Lepêtre-Mouelhi, Sinda; Wack, Séverine; Clayette, Pascal; Rogez-Kreuz, Christine; Yousfi, Rahima; Couvreur, Patrick; Desmaële, Didier

    2010-05-01

    4-(N)-1,1',2-trisnor-squalenoyldideoxycytidine monophosphate (SQddC-MP) and 4-(N)-1,1',2-trisnor-squalenoylgemcitabine monophosphate (SQdFdC-MP) were synthesized using phosphoramidite chemistry. These amphiphilic molecules self-assembled to about hundred nanometers size nanoassemblies in aqueous medium. Nanoassemblies of SQddC-MP displayed significant anti-HIV activity whereas SQdFdC-MP nanoassemblies displayed promising anticancer activity on leukemia cells. These results suggested that squalene conjugate of negatively charged nucleotide analogues efficiently penetrated within cells. Thus, we propose a new prodrug strategy for improved delivery of nucleoside analogues to ameliorate their biological efficacy.

  2. Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway.

    Science.gov (United States)

    Syrovets, T; Tippler, B; Rieks, M; Simmet, T

    1997-06-15

    We have previously reported that the serine protease plasmin generated during contact activation of human plasma triggers biosynthesis of leukotrienes (LTs) in human peripheral monocytes (PMs), but not in polymorphonuclear neutrophils (PMNs). We now show that purified plasmin acts as a potent chemoattractant on human monocytes, but not on PMNs. Human plasmin or plasminogen activated with urokinase, but not active site-blocked plasmin or plasminogen, elicited monocyte migration across polycarbonate membranes. Similarly, stimulation of monocytes with plasmin, but not with active site-blocked plasmin or plasminogen, induced actin polymerization. As assessed by checkerboard analysis, the plasmin-mediated monocyte locomotion was a true chemotaxis. The plasmin-induced chemotactic response was inhibited by the lysine analog trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which prevents binding of plasmin/ogen to the appropriate membrane binding sites. In addition, active site-blocked plasmin inhibited monocyte migration triggered by active plasmin. Further, plasmin-induced monocyte chemotaxis was inhibited by pertussis toxin (PTX) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) and chelerythrine, two structurally unrelated inhibitors of protein kinase C (PKC). Plasmin, but not active site-blocked plasmin or plasminogen, triggered formation of cyclic guanosine monophosphate (cGMP) in monocytes. LY83583, an inhibitor of soluble guanylyl cyclase, inhibited both plasmin-induced cGMP formation and the chemotactic response. The latter effect could be antagonized by 8-bromo-cGMP. In addition, KT5823 and (Rp)-8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate [(Rp)-8-pCPT-cGMPs], two structurally unrelated inhibitors of cGMP-dependent protein kinase, inhibited plasmin-mediated monocyte chemotaxis. Thus, beyond being a stimulus for lipid mediator release, plasmin is a potent and specific chemoattractant for human monocytes acting via a c

  3. Mnk kinase pathway: Cellular functions and biological outcomes

    Institute of Scientific and Technical Information of China (English)

    Sonali; Joshi; Leonidas; C; Platanias

    2014-01-01

    The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.

  4. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    Science.gov (United States)

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

  5. Gold Core Mesoporous Organosilica Shell Degradable Nanoparticles for Two-Photon Imaging and Gemcitabine Monophosphate Delivery

    KAUST Repository

    Rhamani, Saher

    2017-09-12

    The synthesis of gold core degradable mesoporous organosilica shell nanoparticles is described. The nanopaticles were very efficient for two-photon luminescence imaging of cancer cells and for in vitro gemcitabine monophosphate delivery, allowing promising theranostic applications in the nanomedicine field.

  6. DISSIMILARITY IN METHACHOLINE AND ADENOSINE 5'-MONOPHOSPHATE RESPONSIVENESS 3-H AND 24-H AFTER ALLERGEN CHALLENGE

    NARCIS (Netherlands)

    AALBERS, R; KAUFFMAN, HF; KOETER, GH; POSTMA, DS; DEVRIES, K; DEMONCHY, JGR

    1991-01-01

    Bronchial hyperresponsiveness (BHR) to methacholine and adenosine 5'-monophosphate (AMP) was studied in 15 allergic asthmatic patients before and 3 and 24 h after allergen challenge with hose dust mite (HDM). Subjects attended the clinic on 3 consecutive days. On the first day a control solution was

  7. 食管鳞状细胞癌组织中酪氨酸激酶受体B和脑源性神经生长因子蛋白及mRNA的表达及意义%Expression of tyrosine kinase receptorB and brain-derived nenro trophic factor protein and mRNA in esophageal squamous cell carcinoma tissue

    Institute of Scientific and Technical Information of China (English)

    毛红丽; 庞霞; 曲蕴慧

    2012-01-01

    Objective To explore the expressions of tyrosine kinase receptor B (TrkB) and brainderived neuro trophic factor (BDNF) protein and mRNA in esophageal squamous cell carcinoma tissues (ESCC) and their significances.Methods The expressions of TrkB and BDNF protein and mRNA were detected by immunohistochemistry and in situ hybridization in 59 cases of ESCC tissues,27 cases of adjacent atypical hyperplasia epithelium and 36 cases of normal esophageal epithelium.Results The positive rates of TrkB protein and mRNA were 71.2% and 64.4% in ESCC tissues,respectively,which was significantly higher than that in adjacent atypical hyperplasia epithelium (48.1% and 33.3%,respectively) or normal esophageal epithelium tissues ( 0.0% ),and there was a significant difference among the groups (P<0.05).In addition,The positive rates of BDNF protein and mRNA were 76.3% and 69.5% in ESCC tissues,respectively,which was significantly higher than that in adjacent atypical hyperplasia epithelium (55.6% and 40.7%,respectively) or normal esophageal epithelium tissues (0.0% ),and there was a significant difference among the groups ( P < 0.05 ).The expressions of TrkB and BDNF protein and mRNA were closely correlated with differentiation degree,invasion depth and lymph node metastasis in ESCC tissues (P <0.05 ).Further correlation analysis revealed that the expressions of TrkB and BDNF protein and mRNA displayed the positive correlation ( P < 0.05 ).Conclusion TrkB and BDNF protein and mRNA may play a critical role in the metastasis and carcinogenesis of ESCC.%目的 探讨酪氨酸激酶受体B( TrkB)和脑源性神经生长因子(BDNF)蛋白及mRNA在食管鳞状细胞癌(ESCC)组织中的表达及其意义.方法 应用免疫组织化学及原位杂交法检测59例ESCC、27例癌旁不典型增生组织及36例正常食管黏膜组织中TrkB和BDNF蛋白及mRNA的表达.结果 ESCC组织中TrkB蛋白及mRNA的阳性表达率分别为71.2%和64.4%,显著高于

  8. [Age-dependent changes in mRNA transport (nucleus-cytoplasm)].

    Science.gov (United States)

    Müller, W E; Agutter, P S; Prochnow, D J; Fasold, H; Sève, A P; Tsiapalis, C M; Schröder, H C

    1993-01-01

    Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase. In addition, we found that an RNA helicase activity is present in NE, which also may be involved in RNA transport. Our results show that, besides poly(A), also double-stranded RNA structures may modulate RNA export. The amount of mRNA released from nuclei markedly decreases with age. Evidence is presented that this age-dependent change is caused by an impairment of polyadenylation of mRNA, hnRNA processing, release of mRNA from nuclear matrix, and translocations of mRNA from nuclear to cytoplasmic compartment (decrease in activities of NE NTPase, protein kinase, and phosphatase; decrease in poly(A)-binding affinity of mRNA carrier).

  9. Signals fly when kinases meet Rho-of-plants (ROP) small G-proteins.

    Science.gov (United States)

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

    Rho-type small GTP-binding plant proteins function as two-state molecular switches in cellular signalling. There is accumulating evidence that Rho-of-plants (ROP) signalling is positively controlled by plant receptor kinases, through the ROP guanine nucleotide exchange factor proteins. These signalling modules regulate cell polarity, cell shape, hormone responses, and pathogen defence, among other things. Other ROP-regulatory proteins might also be subjected to protein phosphorylation by cellular kinases (e.g., mitogen-activated protein kinases or calcium-dependent protein kinases), in order to integrate various cellular signalling pathways with ROP GTPase-dependent processes. In contrast to the role of kinases in upstream ROP regulation, much less is known about the potential link between ROP GTPases and downstream kinase signalling. In other eukaryotes, Rho-type G-protein-activated kinases are widespread and have a key role in many cellular processes. Recent data indicate the existence of structurally different ROP-activated kinases in plants, but their ROP-dependent biological functions still need to be validated. In addition to these direct interactions, ROPs may also indirectly control the activity of mitogen-activated protein kinases or calcium-dependent protein kinases. These kinases may therefore function as upstream as well as downstream kinases in ROP-mediated signalling pathways, such as the phosphatidylinositol monophosphate kinases involved in cell polarity establishment.

  10. Mutation Study of Two Thymidine Kinases 

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Munch-Petersen, Birgitte; Eklund, Hans

    that phosphorylates all the natural deoxyribonucleosides and like insects, C. elegans only contains a single deoxyribonucleoside kinase-like gene. In contrast to the insects, however, the protein encoded by the elegans gene is 46 % identical to human TK1 (HuTK1) and have no homology to the insect kinase. Like HuTK1...... the C. elegans kinase (CeTK1) has thymidine as the preferred substrate, but it also displays activity with deoxyguanosine, though with high Km. A number of point mutations have been introduced in the active site of both the human and elegans TK's in order to change the substrate specificity away from...... not phosphorylate the anticancer analog 1-β-D-arabinofuranosylcytosine (AraC), however. The HuTK1 mutant has been crystallized, and azidothymidine monophosphate has been modelled into the active site....

  11. Triazole inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    OpenAIRE

    Maurya, Sushil K.; Gollapalli, Deviprasad R.; Kirubakaran, Sivapriya; Zhang, Minjia; Johnson, Corey R.; Benjamin, Nicole N.; Hedstrom, Lizbeth; Gregory D Cuny

    2009-01-01

    Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Since C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole ...

  12. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    Science.gov (United States)

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients.

  13. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    subunits are highly conserved during evolution. The relationship between CK-2 alpha from humans and plants is still 73%. Similar relationships are reported for the beta-subunit. Chromosomal assignment of CK-2 alpha shows two gene loci, one of which is a pseudogene. They are located on different chromosomes......, no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-subunit affecting: (i) stability, (ii) enzyme specificity and (iii) enzyme activity. The question where CK-2 and its subunits are located throughout the cell cycle has also been addressed, mainly because of the large discrepancies that still exist between results obtained by different investigators. Tissue...

  14. Urinary cyclic guanosine 3',5'-monophosphate and cyclic adenosine 3',5'-monophosphate changes in spontaneous and induced onset active labor.

    Science.gov (United States)

    Chen, Da-Chung; Yuan, Shyng-Shiou F; Su, Her-Young; Lo, Shin-Chieh; Ren, Shin-Sia; Wu, Gwo-Jang

    2005-11-01

    The aim of this prospective, randomized study was to investigate the changes in urinary cyclic guanosine 3',5'-monophosphate (cGMP) and cyclic adenosine 3',5'-monophosphate (cAMP) between the latent and the active phases of spontaneous and prostaglandin E(1) (PGE(1))-induced labor. Seventy singleton pregnant women at 36-41(+) weeks' gestation without signs of fetal distress were enrolled. The first group consisted of 35 pregnant women in whom labor was induced by PGE(1) applied intravaginally. The second group consisted of 35 women who had spontaneous active labor. Clinical data of the two groups were assessed as labor progressed. After the onset of active labor, urinary cGMP/creatinine (U cGMP/Cr) decreased in both groups with the percentage decline of 35.2 and 9.7, respectively, but this difference was only significant in the PGE(1)-induced group (P=0.033). After the onset of active labor, urinary cAMP/creatinine (U cAMP/Cr) decreased in both groups with the percentage decline of 36.5 and 15.6, respectively, but this difference was only significant in the PGE(1)-induced group (P=0.001). The duration of the latent phase was significantly shortened in the PGE(1)-induced group compared with the spontaneous labor group (Plabor. Our results suggest that U cGMP/Cr and U cAMP/Cr can serve as easily obtained secondary messenger markers of myometrial contractility and cervical ripening at the onset of active labor. The NO-cGMP system and the G-protein alpha-cAMP system in the human uterus may concomitantly contribute to uterine quiescence during pregnancy and show downregulation in U cGMP/Cr and U cAMP/Cr at the initiation of active labor.

  15. cyclic monophosphate

    African Journals Online (AJOL)

    Administrator

    2006-10-02

    Oct 2, 2006 ... Second messengers are small transient molecules that transmit and/or modulate environmental or hormonal signals ... cyclases (pGCs), and soluble cytosolic guanylyl cyclases ... Figure 2. Model of cGMP generation and cGMP dependent cellular effects. ..... dynamics of colonic epithelial proliferation.

  16. (−-Epicatechin-3-O-β-d-allopyranoside from Davallia formosana, Prevents Diabetes and Hyperlipidemia by Regulation of Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Chun-Ching Shih

    2015-10-01

    Full Text Available The purpose of this experiment was to determine the antidiabetic and lipid-lowering effects of (−-epicatechin-3-O-β-d-allopyranoside (BB from the roots and stems of Davallia formosana in mice. Animal treatment was induced by high-fat diet (HFD or low-fat diet (control diet, CD. After eight weeks of HFD or CD exposure, the HFD mice were treating with BB or rosiglitazone (Rosi or fenofibrate (Feno or water through gavage for another four weeks. However, at 12 weeks, the HFD-fed group had enhanced blood levels of glucose, triglyceride (TG, and insulin. BB treatment significantly decreased blood glucose, TG, and insulin levels. Moreover, visceral fat weights were enhanced in HFD-fed mice, accompanied by increased blood leptin concentrations and decreased adiponectin levels, which were reversed by treatment with BB. Muscular membrane protein levels of glucose transporter 4 (GLUT4 were reduced in HFD-fed mice and significantly enhanced upon administration of BB, Rosi, and Feno. Moreover, BB treatment markedly increased hepatic and skeletal muscular expression levels of phosphorylation of AMP-activated (adenosine monophosphate protein kinase (phospho-AMPK. BB also decreased hepatic mRNA levels of phosphenolpyruvate carboxykinase (PEPCK, which are associated with a decrease in hepatic glucose production. BB-exerted hypotriglyceridemic activity may be partly associated with increased mRNA levels of peroxisome proliferator activated receptor α (PPARα, and with reduced hepatic glycerol-3-phosphate acyltransferase (GPAT mRNA levels in the liver, which decreased triacylglycerol synthesis. Nevertheless, we demonstrated BB was a useful approach for the management of type 2 diabetes and dyslipidemia in this animal model.

  17. Gas-phase spectroscopy of protonated adenine, adenosine 5′-monophosphate and monohydrated ions

    DEFF Research Database (Denmark)

    Pedersen, S.O.; Støchkel, K.; Byskov, C.S.

    2013-01-01

    Microsolvation of chromophore ions commonly has large effects on their electronic structure and as a result on their optical absorption spectra. Here spectroscopy of protonated adenine (AdeH+) and its complex with one water molecule isolated in vacuo was done using a home-built mass spectrometer...... in combination with a tuneable pulsed laser system. Experiments also included the protonated adenosine 5′-monophosphate nucleotide (AMPH+). In the case of bare AdeH+ ions, one-photon absorption leads to four dominant fragment ions corresponding to ammonium and ions formed after loss of either NH3, HCN, or NH2CN...

  18. [Successful treatment of T-cell prolymphocytic leukemia (T-PLL) with fludarabine monophosphate].

    Science.gov (United States)

    Maeda, Akinori; Iwai, Kazuya; Ishibashi, Takafumi

    2009-08-01

    We report a 79-year-old woman with T-cell prolymphocytic leukemia (T-PLL) who was successfully treated with fludarabine monophosphate. She was admitted to our hospital because of dyspnea on effort. On admission, anemia and hepatosplenomegaly were apparent but lymphadenopathy was absent. Peripheral blood examination showed anemia and leukocytosis with 29.5% abnormal lymphocytes. The bone marrow was infiltrated with 84.1% abnormal lymphocytes. The nucleolus was visible in some of these abnormal cells. These cells were positive for CD2, CD3, CD4, CD5, CD7, CD38, CD52, and negative for CD8, CD10, CD19, CD20, CD25, CD56. Based on these findings, she was diagnosed as having T-PLL. Therapy with oral cyclophosphamide (50 mg/day) was started, but was discontinued because of agranulocytosis. Then, she received intravenous fludarabine monophosphate (30 mg/day) on days 1-5 every four to five weeks. The reticulocyte count increased gradually, and she became free from red cell transfusions. Unfortunately, she finally died from massive gastro intestinal hemorrhage, but T-PLL was well controlled at the time of death.

  19. The antilipolytic agent 3,5-dimethylpyrazole inhibits insulin release in response to both nutrient secretagogues and cyclic adenosine monophosphate agonists in isolated rat islets.

    Science.gov (United States)

    Masiello, P; Novelli, M; Bombara, M; Fierabracci, V; Vittorini, S; Prentki, M; Bergamini, E

    2002-01-01

    This study intended to test the hypothesis that intracellular lipolysis in the pancreatic beta cells is implicated in the regulation of insulin secretion stimulated by nutrient secretagogues or cyclic adenosine monophosphate (cAMP) agonists. Indeed, although lipid signaling molecules were repeatedly reported to influence beta-cell function, the contribution of intracellular triglycerides to the generation of these molecules has remained elusive. Thus, we have studied insulin secretion of isolated rat pancreatic islets in response to various secretagogues in the presence or absence of 3,5-dimethylpyrazole (DMP), a water-soluble and highly effective antilipolytic agent, as previously shown in vivo. In vitro exposure of islets to DMP resulted in an inhibition (by approximately 50%) of the insulin release stimulated not only by high glucose, but also by another nutrient secretagogue, 2-ketoisocaproate, as well as the cAMP agonists 3-isobutyl-1-methylxanthine and glucagon. The inhibitory effect of DMP, which was not due to alteration of islet glucose oxidation, could be reversed upon addition of sn-1,2-dioctanoylglycerol, a synthetic diglyceride, which activates protein kinase C. The results provide direct pharmacologic evidence supporting the concept that endogenous beta-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists.

  20. Icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels in the hippocampus of the senescence- accelerated mouse

    Institute of Scientific and Technical Information of China (English)

    Zhanwei Zhang; Ting Zhang; Keli Dong

    2012-01-01

    At 8 weeks after intragastric administration of icariin to senescence-accelerated mice (P8 strain), Morris water maze results showed that escape latency was shortened, and the number of platform crossings was increased. Immunohistochemical staining and western blot assay detected signifi-cantly increased levels of cyclic adenosine monophosphate response element binding protein. These results suggest that icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels and improves learning and memory functions in hippo-campus of the senescence-accelerated mouse.

  1. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients

    OpenAIRE

    Sasaki, Hidefumi; SHITARA, MASAYUKI; YOKOTA, KEISUKE; OKUDA, KATSUHIRO; HIKOSAKA, YU; MORIYAMA, SATORU; YANO, MOTOKI; Fujii, Yoshitaka

    2012-01-01

    Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or abs...

  2. Osmium (VI) complexes of the 3', 5'-dinucleoside monophosphates, ApU and UpA.

    Science.gov (United States)

    Daniel, F B; Behrman, E J

    1976-02-10

    The dinucleoside monophosphates, ApU and UpA, react with potassium osmate (VI) and 2,2'-bipyridyl to form the corresponding oxo-osmium (VI) bipyridyl sugar ester in which the osmate group is bonded to the terminal 2',3'-glycol. Osmium (VIII) tetroxide and 2,2'-bipyridyl react with the dinucleosides to form the corresponding oxo-osmium (VI) bipyridyl heterocyclic esters which result from addition of the tetroxide to the 5,6-double bond of the uracil residue. Although capable of transesterification reactions, these heterocyclic esters are exceptionally stable toward exchange reactions in solution. No apparent exchange was observed after 1 month. This reaction thus seems promising for single-site osmium labeling in polynucleotides.

  3. Influences of dibutyryl cyclic adenosine monophosphate and forskolin on human sperm motility in vitro

    Institute of Scientific and Technical Information of China (English)

    Ji-HongLIU; YangLI; Zheng-GuoCAO; Zhang-QunYE

    2003-01-01

    Aim: To study the influences of dibutyryl cyclic adenosine monophosphate (dbcAMP) and forskolin on human sperm motility in vitro. Methods: Semen samples, aseptically obtained by masturbation and prepared by swim-up technique from 20 fertile men, were incubated with different concenlrations of dbcAMP and forskolin at 37℃. Measurements were carried out after l0 min, 20 min, 30 min and 60 min incubation. Motility parameters were estimated by using an automatic analyzing system. Results: Treatment with dbcAMP or forskolin resulted in a significant increase in sperm motility and progressive motility. The larger the concenlrations of dbcAMP or forskolin,the greater the effect appeared. The straight linear velocity and curvilinear velocity were not affected by both agents.Conclusion: dbcAMP and forskolin increase the motility and progressive motility of human sperm in vitro. ( Asian J Androl 2003 Jun; 5: 113-115)

  4. Corticosteroid-Responsive Pulmonary Toxicity Associated with Fludarabine Monophosphate: A Case Report

    Directory of Open Access Journals (Sweden)

    Milda Rudzianskiene

    2012-12-01

    Full Text Available Fludarabine monophosphate is an effective drug for the treatment of lymphoid malignancies. Myelosuppression, opportunistic infections, and autoimmune hemolytic anemia are the most common side effects of fludarabine. Herein we report a 55-year-old female that presented with fever and dyspnea after completing her third cycle of FMD (fludarabine, mitoxantrone, and dexamethasone chemotherapy for stage IV non-Hodgkin follicular lymphoma. Chest X-ray revealed bilateral pneumofibrotic changes and chest CT showed bilateral diffuse interstitial changes with fibrotic alterations. No evidence of infectious agents was noted. The patient had a reduced carbon monoxide transfer factor (45%. Her symptoms and radiographic findings resolved following treatment with prednisolone. The literature contains several cases of fludarabine-associated interstitial pulmonary toxicity that responded to steroid therapy. Fludarabine-induced pulmonary toxicity is reversible with cessation of the drug and administration of glucocorticosteroids.

  5. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P. (TGRI); (Toronto)

    2010-06-14

    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  6. Enhanced tumor necrosis factor suppression and cyclic adenosine monophosphate accumulation by combination of phosphodiesterase inhibitors and prostanoids

    NARCIS (Netherlands)

    Sinha, B; Semmler, J; Eisenhut, T; Eigler, A; Endres, S

    1995-01-01

    We investigated cooperative effects of phosphodiesterase (PDE) inhibitors and prostanoids on cyclic adenosine monophosphate (cAMP) accumulation and tumor necrosis factor (TNF)-alpha synthesis in human peripheral blood mononuclear cells (PBMC). PDE inhibitors alone induced only a small increase in cA

  7. Protective effect of oral terfenadine and not inhaled ipratropium on adenosine 5 '-monophosphate-induced bronchoconstriction in patients with COPD

    NARCIS (Netherlands)

    Rutgers, [No Value; Koeter, GH; Van der Mark, TW; Postma, DS

    1999-01-01

    Background Inhalation of adenosine 5'-monophosphate (AMP) causes bronchoconstriction in patients with asthma and in many patients with chronic obstructive pulmonary disease (COPD). In asthma, AMP-induced bronchoconstriction has been shown to be determined mainly by release of mast cell mediators, an

  8. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

    Science.gov (United States)

    Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

    2015-10-01

    This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

  9. Expression of casein kinase 2 during mouse embryogenesis

    DEFF Research Database (Denmark)

    Mestres, P; Boldyreff, B; Ebensperger, C;

    1994-01-01

    This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level by immunohisto......This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level...

  10. Relationship Between Heart Damages and HSPs mRNA in Persistent Heat Stressed Broilers

    Institute of Scientific and Technical Information of China (English)

    SUN Pei-ming; LIU Yu-tian; ZHAO Yong-gang; BAO En-dong; WANG Zhi-liang

    2007-01-01

    The relationship between myocardial cell damages and HSPs mRNA transcription in heat stressed broilers was studied using a spectrophotometer, the histopathological technique, and fluorescence quantitative reverse transcription PCR (FQ RT-PCR). The results showed that the activities of creatine kinase (CK) and glutamic-pyruvic transaninase (GPT) were induction during the persistent heat stress. The major lesions of the myocardial fibers were granular degeneration and necrosis. The transcription of constitutive or cognate heat shock protein 70 (HSC70) mRNA was changeable. The transcription of heat shock protein 70 (HSP70) mRNA was increased obviously in the course of persistent heat stress. The results showed that the change of HSC70 mRNA transcription was contrary to the activity of CK, and the level of HSC70 mRNA transcription must be used as a symbol of the myocardial cell damages in the course of persistent heat stress.

  11. Domain compatibility in Ire1 kinase is critical for the Unfolded Protein Response

    OpenAIRE

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J.; Tirasophon, Witoon

    2010-01-01

    The unfolded phrotein response is a mechanism to cope with endoplasmic reticulum stress. In Saccharomyces cerevisiae, Ire1 senses the stress and mediates a signaling cascade to upregulate responsive genes through an unusual HAC1 mRNA splicing. The splicing requires interconnected activity (kinase and endoribonuclease) of Ire1 to cleave HAC1 mRNA at the non-canonical splice sites before translation into Hac1 transcription factor. Analysis of the truncated kinase domain from Ire1 homologs revea...

  12. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  13. Promotion and inhibition of cardiac hypertrophy by A-kinase anchor proteins.

    Science.gov (United States)

    Blant, Alexandra; Czubryt, Michael P

    2012-09-01

    Originally identified as mediators of cyclic adenosine monophosphate (cAMP) and protein kinase A signaling, A-kinase anchor proteins (AKAPs) are now recognized as a diverse family of molecular scaffolds capable of interacting with many other proteins. Members of the AKAP family within the heart can take on either pro- or anti-hypertrophic roles by interacting with a myriad of protein kinases and phosphatases in the process. AKAPs often form the core of large signaling complexes (or signalosomes) that allow multiple pathways to converge and functionally intertwine. Approximately 30% of AKAPs discovered to date are expressed in the heart, but the functions of many of these remain to be discovered. This review focuses on AKAPs that have been demonstrated to play roles in mediating cardiac hypertrophy.

  14. Control of meristem development by CLAVATA1 receptor kinase and kinase-associated protein phosphatase interactions

    Energy Technology Data Exchange (ETDEWEB)

    Stone, J.M.; Walker, J.C. [Univ. of Missouri, Columbia, MO (United States). Div. of Biological Sciences; Trotochaud, A.E.; Clark, S.E. [Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Biology

    1998-08-01

    The CLAVATA1 (CLV1) gene encodes a putative receptor kinase required for the proper balance between cell proliferation and differentiation in Arabidopsis shoot and flower meristems. Impaired CLV1 signaling results in masses of undifferentiated cells at the shoot and floral meristems. Although many putative receptor kinases have been identified in plants, the mechanism of signal transduction mediated by plant receptor-like kinases is largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that binds to multiple plant receptor-like kinases in a phosphorylation-dependent manner. To examine a possible role for KAPP in CLV1-dependent plant development, the interaction of CLV1 and KAPP was investigated in vitro and in vivo. KAPP binds directly to autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in plant extracts derived from meristematic tissue. Reduction of KAPP transcript accumulation in an intermediate clv1 mutant suppresses the mutant phenotype, and the degree of suppression is inversely correlated with KAPP mRNA levels. These data suggest that KAPP functions as a negative regulator of CLV1 signaling in plant development. This may represent a general model for the interaction of KAPP with receptor kinases.

  15. Altered Regulation of Protein Kinase A Activity in the Medial Prefrontal Cortex of Normal and Brain-Injured Animals Actively Engaged in a Working Memory Task

    OpenAIRE

    Kobori, Nobuhide; Moore, Anthony N.; Pramod K Dash

    2015-01-01

    Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is required for short- and long-term memory. In contrast, enhanced PKA activity has been shown to impair working memory, a prefrontal cortex (PFC)-dependent, transient form of memory critical for cognition and goal-directed behaviors. Working memory can be impaired after traumatic brain injury (TBI) in the absence of overt damage to the PFC. The cellular and molecular mechanisms that contribute to this deficit ar...

  16. 微小RNA-155对内毒素血症幼鼠肝脏白细胞介素-1受体相关激酶-1、4mRNA表达的影响%Effects of microRNA-155 on interleukin-1 receptor-associated kinase 1 and 4 mRNA expression in liver injury of endotoximia mice

    Institute of Scientific and Technical Information of China (English)

    吕鑫; 张育才; 崔云; 任玉倩; 李芮; 戎群芳

    2015-01-01

    目的 探讨微小RNA-155(microRNA-155,miRNA-155)对内毒素血症幼年小鼠肝组织白细胞介素(interleukin,IL)-1受体相关激酶(interleukin-1 receptor-associated kinase,IRAK)-1 mRNA及 IRAK-4 mRNA表达的影响.方法 120只3~5周龄雄性BALB/c小鼠,随机数字表法随机分为内毒素组、miRNA-155抑制物组和对照组,每组各40只.miRNA-155抑制物组注射内毒素前于尾静脉注射miRNA-155抑制物180mg/(kg·d).内毒素组和miRNA-155抑制物组腹腔注射内毒素20 mg/kg,对照组腹腔注射等容量生理盐水.注射内毒素后6、12、24、48 h(每亚组各10只)分别处死,留取肝脏组织标本.实时荧光定量PCR法检测肝组织miRNA-155、IRAK-1 mRNA、IRAK-4 mRNA表达,ELISA法测定肝组织肿瘤坏死因子(tumor necrosis factor,TNF)-α、IL-1和IL-10的表达;观察肝组织病理变化.结果 内毒素组及miRNA-155抑制物组小鼠肝脏miRNA-155表达较对照组升高,在6h达峰值,后逐渐下降,在48 h各组水平趋于一致,各组间比较在6h、12 h、24 h差异有统计学意义(P<0.05).内毒素组及miRNA-155抑制物组IRAK-1 mRNA及IRAK-4 mRNA表达较对照组水平升高,miRNA-155抑制物组较内毒素组降低,12、24、48 h时间点,3组间小鼠肝组织IRAK-1 mRNA及IRAK-4 mRNA表达水平的差异有统计学意义(P<0.05).内毒素组及miRNA-155抑制物组肝组织TNF-α、IL-1、IL-10水平较对照组升高,miRNA-155抑制物组较内毒素组TNF-α、IL-1、IL-10水平下降,各组间比较差异有统计学意义(P<0.05).HE染色下观察肝组织病变:内毒素组小鼠肝组织病理损伤出现早,损伤程度重,miRNA-155抑制物组病理损伤程度相对较轻.结论 抑制内毒素血症小鼠miRNA-155,小鼠肝组织IRAK-1 mRNA及IRAK-4 mRNA表达下降,炎症因子水平下降,肝脏病理损伤减轻.%Objective To explore the protective effect of rnicroRNA (miRNA)-155 inhibitor on interleukin-1 receptor-associated kinase (IRAK)-1 mRNA and IRAK-4 mRNA

  17. Effects of metformin on expression of AMP-activated protein kinase in rat glomerular mesangial cells

    Institute of Scientific and Technical Information of China (English)

    顾俊菲

    2014-01-01

    Objective To observe the effects of metformin on expression of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),nuclear factor-κB(NF-κB)and transforming growth factorβ1(TGF-β1)in cultured rat glomerular mesangial cells(MCs),and explore its reno-protective mechanisms.Methods MCs were cultured in the medium with normal glucose(group NG,5.6mmol/L),high glucose(group HG,25 mmol/L)and different concentrations of metformin(group M1,M2,M3).After 48 h exposure,the supernatants and MCs

  18. Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

    OpenAIRE

    Rattan, R; Giri, S.; Hartmann, LC; Shridhar, V

    2009-01-01

    Abstract Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α1, -β1, -γ1 and -γ2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian ca...

  19. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs.

    Science.gov (United States)

    Vlasova-St Louis, Irina; Bohjanen, Paul R

    2016-01-25

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP.

  20. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs

    Directory of Open Access Journals (Sweden)

    Irina Vlasova-St. Louis

    2016-01-01

    Full Text Available In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE binding proteins, and the GU-rich element (GRE binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP.

  1. The AreA transcription factor mediates the regulation of deoxynivalenol (DON) synthesis by ammonium and cyclic adenosine monophosphate (cAMP) signalling in Fusarium graminearum.

    Science.gov (United States)

    Hou, Rui; Jiang, Cong; Zheng, Qian; Wang, Chenfang; Xu, Jin-Rong

    2015-12-01

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium graminearum, is harmful to humans and animals. Because different nitrogen sources are known to have opposite effects on DON production, in this study, we characterized the regulatory mechanisms of the AREA transcription factor in trichothecene biosynthesis. The ΔareA mutant showed significantly reduced vegetative growth and DON production in cultures inoculated with hyphae. Suppression of TRI gene expression and DON production by ammonium were diminished in the ΔareA mutant. The deletion of AREA also affected the stimulatory effects of arginine on DON biosynthesis. The AreA-green fluorescent protein (GFP) fusion complemented the ΔareA mutant, and its localization to the nucleus was enhanced under nitrogen starvation conditions. Site-directed mutagenesis showed that the conserved predicted protein kinase A (PKA) phosphorylation site S874 was important for AreA function, indicating that AreA may be a downstream target of the cyclic adenosine monophosphate (cAMP)-PKA pathway, which is known to regulate DON production. We also showed that AreA interacted with Tri10 in co-immunoprecipitation assays. The interaction of AreA with Tri10 is probably related to its role in the regulation of TRI gene expression. Interestingly, the ΔareA mutant showed significantly reduced PKA activity and expression of all three predicted ammonium permease (MEP) genes, in particular MEP1, under low ammonium conditions. Taken together, our results show that AREA is involved in the regulation of DON production by ammonium suppression and the cAMP-PKA pathway. The AreA transcription factor may interact with Tri10 and control the expression and up-regulation of MEP genes.

  2. Controlled supramolecular structure of guanosine monophosphate in the interlayer space of layered double hydroxide

    Directory of Open Access Journals (Sweden)

    Gyeong-Hyeon Gwak

    2016-12-01

    Full Text Available Guanosine monophosphates (GMPs were intercalated into the interlayer space of layered double hydroxides (LDHs and the molecular arrangement of GMP was controlled in LDHs. The intercalation conditions such as GMP/LDH molar ratio and reaction temperature were systematically adjusted. When the GMP/LDH molar ratio was 1:2, which corresponds to the charge balance between positive LDH sheets and GMP anions, GMP molecules were well-intercalated to LDH. At high temperature (100 and 80 °C, a single GMP molecule existed separately in the LDH interlayer. On the other hand, at lower temperature (20, 40 and 60 °C, GMPs tended to form ribbon-type supramolecular assemblies. Differential scanning calorimetry showed that the ribbon-type GMP assembly had an intermolecular interaction energy of ≈101 kJ/mol, which corresponds to a double hydrogen bond between guanosine molecules. Once stabilized, the interlayer GMP orientations, single molecular and ribbon phase, were successfully converted to the other phase by adjusting the external environment by stoichiometry or temperature control.

  3. Post-translational Analysis of Arabidopsis thaliana Proteins in Response to Cyclic Guanosine Monophosphate Treatment

    KAUST Repository

    Parrott, Brian

    2011-12-12

    The introduction of mass spectrometry techniques to the field of biology has made possible the exploration of the proteome as a whole system as opposed to prior techniques, such as anti-body based assays or yeast two-hybrid studies, which were strictly limited to the study of a few proteins at a time. This practice has allowed for a systems biology approach of exploring the proteome, with the possibility of viewing entire pathways over increments of time. In this study, the effect of treating Arabidopsis thaliana suspension culture cells with 3’,5’-cyclic guanosine monophosphate (cGMP), which is a native second messenger, was examined. Samples were collected at four time points and proteins were extracted and enriched for both oxidation and phosphorylation before analysis via mass spectrometry. Preliminary results suggest a tendency towards an increased number of phosphorylated proteins as a result of cGMP treatment. The data also showed a sharp increase in methionine oxidation in response to the treatment, occurring within the first ten minutes. This finding suggests that cGMP may utilize methionine oxidation as a mechanism of signal transduction. As such, this study corroborates a growing body of evidence supporting the inclusion of methionine oxidation in intracellular signaling pathways.

  4. PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS OF INOSINE MONOPHOSPHATE DEHYDROGENASE (IMPDH) ACTIVITY IN MMF-TREATED HCT RECIPIENTS

    Science.gov (United States)

    Li, Hong; Mager, Donald E.; Sandmaier, Brenda M.; Storer, Barry E.; Boeckh, Michael J.; Bemer, Meagan J.; Phillips, Brian R.; Risler, Linda J.; McCune, Jeannine S.

    2014-01-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplant (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNC) at five time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in the pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic/dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory Emax model with an IC50 = 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, non-relapse mortality, and overall mortality. In conclusion, a pharmacokinetic/dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker. PMID:24727337

  5. Triazole inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    Science.gov (United States)

    Maurya, Sushil K.; Gollapalli, Deviprasad R.; Kirubakaran, Sivapriya; Zhang, Minjia; Johnson, Corey R.; Benjamin, Nicole N.; Hedstrom, Lizbeth; Cuny, Gregory D.

    2010-01-01

    Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Since C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure-activity relationship study revealed that a small alkyl group on the alpha-position of the ether was required with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis. PMID:19624136

  6. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    Science.gov (United States)

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  7. [Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium].

    Science.gov (United States)

    Matveeva, N B; Teplov, V A; Nezvetskiĭ, A R; Orlova, T G; Beĭlina, S I

    2012-01-01

    Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0,1-1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced typical for attractant action the increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.

  8. Ion-exclusion chromatography determination of organic acid in uridine 5'-monophosphate fermentation broth.

    Science.gov (United States)

    Niu, Huanqing; Chen, Yong; Xie, Jingjing; Chen, Xiaochun; Bai, Jianxin; Wu, Jinglan; Liu, Dong; Ying, Hanjie

    2012-09-01

    Simultaneous determination of organic acids using ion-exclusion liquid chromatography and ultraviolet detection is described. The chromatographic conditions are optimized when an Aminex HPX-87H column (300 × 7.8 mm) is employed, with a solution of 3 mmol/L sulfuric acid as eluent, a flow rate of 0.4 mL/min and a column temperature of 60°C. Eight organic acids (including orotic acid, α-ketoglutaric acid, citric acid, pyruvic acid, malic acid, succinic acid, lactic acid and acetic acid) and one nucleotide are successfully quantified. The calibration curves for these analytes are linear, with correlation coefficients exceeding 0.999. The average recovery of organic acids is in the range of 97.6% ∼ 103.1%, and the relative standard deviation is in the range of 0.037% ∼ 0.38%. The method is subsequently applied to obtain organic acid profiles of uridine 5'-monophosphate culture broth fermented from orotic acid by Saccharomyces cerevisiae. These data demonstrate the quantitative accuracy for nucleotide fermentation mixtures, and suggest that the method may also be applicable to other biological samples.

  9. Cyclic adenosine 3',5'-monophosphate and germination of sporangiospores from the fungus Mucor.

    Science.gov (United States)

    Orlowski, M

    1980-06-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.

  10. The clinical correlation of regulatory T cells and cyclic adenosine monophosphate in enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Shih-Min Wang

    Full Text Available Brainstem encephalitis (BE and pulmonary edema (PE are notable complications of enterovirus 71 (EV71 infection.This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD or BE group, and the autonomic nervous system (ANS dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP levels, and the regulatory T cell (Tregs profiles of the patients were determined.Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4(+CD25(+Foxp3+ and CD4(+Foxp3(+ T cells compared with patients with HFMD or BE. The expression frequency of CD4-CD8- was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4+Foxp3+ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.

  11. Lymphocyte beta 2-adrenoceptors and adenosine 3':5'-cyclic monophosphate during and after normal pregnancy.

    Science.gov (United States)

    von Mandach, U.; Gubler, H. P.; Engel, G.; Huch, R.; Huch, A.

    1993-01-01

    1. The beta 2-sympathomimetics, used to inhibit preterm labour, bind predominantly to beta 2-adrenoceptors, activating adenylate cyclase to form adenosine 3':5'-cyclic monophosphate (cyclic AMP), a messenger substance which inhibits the enzyme cascade triggering smooth muscle contraction. beta 2-Adrenoceptor density and cyclic AMP formation can be used as markers of beta 2-adrenergic effect. 2. The present study addresses the influence of pregnancy on the beta-adrenoceptor system. beta 2-Adrenoceptor density and cyclic AMP concentrations (basal and evoked by isoprenaline) in circulating lymphocytes were determined at three points in gestation (16, 29 and 37 weeks) and 9 weeks post partum in 22 normal pregnancies. (-)-[125Iodo]-cyanopindolol was used as the ligand to identify a homogeneous population of beta 2-adrenoceptors on lymphocytes. B- and T-cell fractions were estimated from the same samples. 3. beta 2-Adrenoceptor density decreased significantly during gestation until week 37 (P < 0.01), then increased post partum (P < 0.005). Cyclic AMP concentrations (basal and evoked by isoprenaline) were significantly lower after 16 weeks of gestation than post partum (P < 0.05). 4. The results, which cannot be explained in terms of a shift in the lymphocyte (B- and T-cell) ratio, indicate that beta-adrenoceptor density and function are reduced in normal pregnancy and only return to normal post partum. These findings may be of significance in devising future tocolytic therapy with beta 2-adrenoceptor agonists. PMID:8383562

  12. Lymphocyte beta 2-adrenoceptors and adenosine 3':5'-cyclic monophosphate during and after normal pregnancy.

    Science.gov (United States)

    von Mandach, U; Gubler, H P; Engel, G; Huch, R; Huch, A

    1993-02-01

    1. The beta 2-sympathomimetics, used to inhibit preterm labour, bind predominantly to beta 2-adrenoceptors, activating adenylate cyclase to form adenosine 3':5'-cyclic monophosphate (cyclic AMP), a messenger substance which inhibits the enzyme cascade triggering smooth muscle contraction. beta 2-Adrenoceptor density and cyclic AMP formation can be used as markers of beta 2-adrenergic effect. 2. The present study addresses the influence of pregnancy on the beta-adrenoceptor system. beta 2-Adrenoceptor density and cyclic AMP concentrations (basal and evoked by isoprenaline) in circulating lymphocytes were determined at three points in gestation (16, 29 and 37 weeks) and 9 weeks post partum in 22 normal pregnancies. (-)-[125Iodo]-cyanopindolol was used as the ligand to identify a homogeneous population of beta 2-adrenoceptors on lymphocytes. B- and T-cell fractions were estimated from the same samples. 3. beta 2-Adrenoceptor density decreased significantly during gestation until week 37 (P < 0.01), then increased post partum (P < 0.005). Cyclic AMP concentrations (basal and evoked by isoprenaline) were significantly lower after 16 weeks of gestation than post partum (P < 0.05). 4. The results, which cannot be explained in terms of a shift in the lymphocyte (B- and T-cell) ratio, indicate that beta-adrenoceptor density and function are reduced in normal pregnancy and only return to normal post partum. These findings may be of significance in devising future tocolytic therapy with beta 2-adrenoceptor agonists.

  13. AMP-activated protein kinase: An emerging target for ginseng

    Directory of Open Access Journals (Sweden)

    Kyong Ju Jeong

    2014-04-01

    Full Text Available The adenosine monophosphate (AMP-activated protein kinase (AMPK is a key sensor of cellular energy. Once activated, it switches on catabolic pathways generating adenosine triphosphate (ATP, while switching off biosynthetic pathways consuming ATP. Pharmacological activation of AMPK by metformin holds a therapeutic potential to reverse metabolic abnormalities such as type 2 diabetes and nonalcoholic fatty liver disease. In addition, altered metabolism of tumor cells is widely recognized and AMPK is a potential target for cancer prevention and/or treatment. Panax ginseng is known to be useful for treatment and/or prevention of cancer and metabolic diseases including diabetes, hyperlipidemia, and obesity. In this review, we discuss the ginseng extracts and ginsenosides that activate AMPK, we clarify the various mechanisms by which they achieve this, and we discuss the evidence that shows that ginseng or ginsenosides might be useful in the treatment and/or prevention of metabolic diseases and cancer.

  14. Structure-Based Design, Synthesis, Evaluation And Crystal Structures of Transition State Analogue Inhibitors of Inosine Monophosphate Cyclohydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Xu, L.; Chong, Y.; Hwang, I.; D' Onofrio, A.; Amore, K.; Beardsley, G.P.; Li, C.; Olson, A.J.; Boger, D.L.; Wilson, I.A.; /Skaggs Inst. Chem. Biol. /Scripps Res. Inst.

    2007-07-13

    The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATIC in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.

  15. Predictive value of adenosine 5'-monophosphate challenge in preschool children for the diagnosis of asthma 5 years later.

    Science.gov (United States)

    Cohen, Shlomo; Avital, Avraham; Hevroni, Avigdor; Avenshtein, Alina; Hadi, Ronen; Springer, Chaim

    2012-07-01

    We evaluated the predictive values of preschool bronchial challenge with nebulized adenosine 5'-monophosphate (AMP) using the auscultation method for having asthma 5 years later. Preschool AMP challenge had a high negative (90%) and a moderate positive (67%) predictive value for asthma 5 years later. Positive predictive value increased with the age at which the challenge was performed. The degree of preschool response to AMP was associated with the severity of asthma at school age.

  16. Effect of Sodium-Potassium Pump Inhibitors and Membrane-Depolarizing Agents on Sodium Nitroprusside-Induced Relaxation and Cyclic Guanosine Monophosphate Accumulation in Rat Aorta

    National Research Council Canada - National Science Library

    Rapoport, Robert M; Schwartz, Karen; Murad, Ferid

    1985-01-01

    ... or tetraethylammonium, membrane-depolarizing agents, inhibited relaxation to nitroprusside. These conditions had little or no effect on the elevated cyclic guanosine monophosphate levels at a concentration of nitroprusside (0.1 μM...

  17. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon; (UIC); (MXPL-G)

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  18. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    Science.gov (United States)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  19. Main: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 S000069 17-May-1998 (last modified) kehi Cis element in 3' end region ...of wheat (T.a.) histone H3 mRNA; 3' end formation; Also found in histone genes of other plants, yeast, etc; histone H3; mRNA

  20. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients.

    Science.gov (United States)

    Sasaki, Hidefumi; Shitara, Masayuki; Yokota, Keisuke; Okuda, Katsuhiro; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka

    2012-07-01

    Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or absence of DDR2 mutations at the kinase and discoidin domain was analyzed by direct sequencing. In this cohort, DDR2 mutations were not observed in the 166 patients with lung cancer, although DDR2 polymorphisms were observed (H136H, n=14) at the discoidin domain. mRNA levels of DDR2 in lung tumor samples and the adjacent normal lung samples were simultaneously analyzed. DDR2 mRNA levels were significantly decreased in tumor samples compared with normal lung samples. However, the DDR2 mRNA levels were elevated in the DDR2 polymorphism cases.

  1. cGMP-Dependent Protein Kinase Type I Is Implicated in the Regulation of the Timing and Quality of Sleep and Wakefulness

    OpenAIRE

    Sonja Langmesser; Paul Franken; Susanne Feil; Yann Emmenegger; Urs Albrecht; Robert Feil

    2009-01-01

    Many effects of nitric oxide (NO) are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP). cGMP activates cGMP-dependent protein kinases (PRKGs), which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1) ...

  2. Protein Kinase N2 Regulates AMP-Kinase Signaling and Insulin Responsiveness of Glucose Metabolism in Skeletal Muscle.

    Science.gov (United States)

    Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

    2017-07-18

    Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. As skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. While Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, while stimulating fatty acid oxidation and incorporation into triglycerides, and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC1α and SREBP1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017, American Journal of Physiology-Endocrinology and Metabolism.

  3. BUB1 mRNA is significantly co-expressed with AURKA and AURKB mRNA in advanced-stage ovarian serous carcinoma.

    Science.gov (United States)

    Davidson, Ben; Nymoen, Dag Andre; Elgaaen, Bente Vilming; Staff, Anne Cathrine; Tropé, Claes G; Kærn, Janne; Reich, Reuven; Falkenthal, Thea E Hetland

    2014-06-01

    The objective of this study was to investigate the expression and clinical role of the spindle checkpoint kinase budding uninhibited by benzimidazole 1 (Bub1) in primary and metastatic advanced-stage ovarian serous carcinoma. BUB1 mRNA expression was analyzed in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction (PCR). Bub1 protein expression by Western blotting was studied in 63 carcinomas (30 effusions and 33 solid lesions). BUB1 mRNA expression at different anatomic sites was studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. BUB1 mRNA was universally expressed in serous carcinomas, irrespective of anatomic site. BUB1 mRNA levels were uniformly low in six ovarian surface epithelium specimens analyzed for comparative purposes. Bub1 protein was expressed in 22/30 effusions and 28/33 solid lesions. BUB1 mRNA expression was significantly higher in chemo-naïve primary carcinomas and solid metastases compared to specimens obtained following neoadjuvant chemotherapy (p cancer. BUB1 mRNA levels are lower following chemotherapy exposure in solid lesions, though its presence is unrelated to clinical behavior including response to chemotherapy and survival. BUB1 is co-expressed with AURKA and AURKB suggesting biological relationship between these spindle cell components.

  4. Association between plasma cyclic guanosine monophosphate levels and hemodynamic instability during liver transplantation.

    Science.gov (United States)

    Bezinover, Dmitri; Kadry, Zakiyah; Uemura, Tadahiro; Sharghi, Michael; Mastro, Andrea M; Sosnoski, Donna M; Dalal, Priti; Janicki, Piotr K

    2013-02-01

    The activation of cyclic guanosine monophosphate (cGMP) production in patients with end-stage liver disease (ESLD) has been associated with hemodynamic instability during orthotopic liver transplantation (OLT). The aim of this prospective, observational study was to investigate the involvement of cGMP in the mediation of profound hypotension during liver graft reperfusion. An additional objective was to determine whether preoperative cGMP levels are associated with intraoperative hemodynamic instability. Forty-four consecutive patients undergoing OLT were included in the study. Blood samples for cGMP analysis were obtained from (1) the radial artery before the surgical incision; (2) the radial artery, portal vein, and flush blood during the anhepatic phase; and (3) the radial artery 20 minutes after liver graft reperfusion. On the basis of a statistical analysis, the patients were divided into 2 groups: group 1 (preoperative cGMP level ≥ 0.05 μmol/L) and group 2 (preoperative cGMP level < 0.05 μmol/L). We demonstrated a significant correlation between the preoperative levels of cGMP and the amount of catecholamine required to maintain hemodynamic stability during reperfusion (r = 0.52, P < 0.001), the length of the hospital stay (r = 0.38, P = 0.01), and the length of the intensive care unit (ICU) stay (r = 0.44, P = 0.004). We also demonstrated a significantly higher intraoperative catecholamine requirement (P < 0.001) and a prolonged postoperative ICU stay (P = 0.02) in group 1 patients versus group 2 patients. In conclusion, this study demonstrates increased baseline cGMP production in patients with ESLD, which is significantly associated with severe hypotension during OLT. We suggest that preoperative levels of cGMP correlate with hemodynamic instability during liver graft reperfusion. Copyright © 2012 American Association for the Study of Liver Diseases.

  5. Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation

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    S. Yeswanth

    2013-10-01

    Full Text Available Background: In Staphylococcus aureus purine metabolism plays a crucial role in the formation of biofilm which is a key pathogenic factor. The present study is aimed in the characterization of inosine monophosphate dehydrogenase (IMPDH from Staphylococcus aureus ATCC 12600. Methods: IMPDH gene was amplified using primers designed from IMPDH gene sequence of S. aureus reported in the database. Then polymerase chain reaction (PCR product was cloned in the Sma I site of M13mp18 and expressed in Escherichia coli JM109. The recombinant IMPDH (rIMPDH was overexpressed with 1 mM isopropyl beta-D-1- thiogalactopyranoside (IPTG; Michaelis constant (Km, maximum enzyme velocity (Vmax and catalytic constant (Kcat of expressed IMPDH were determined. Results: The enzyme kinetics of IMPDH grown under aerobic conditions showed a Km of 43.71±1.56 µM, Vmax of 0.247±0.84/µM/mg/min and Kcat of 2.74±0.015/min while in anaerobic conditions the kinetics showed Km of 42.81±3.154/ µM, Vmax of 0.378±0.036 µM/mg/min and Kcat of 4.78±0.021 /min, indicating elevated levels of IMPDH activity under anaerobic conditions. Three-folds increased activity in the presence of 1 mM adenosine triphosphate (ATP correlated with biofilm formation. The kinetics of pure rIMPDH were close to the native IMPDH of S. aureus ATCC12600 and the enzyme showed single band in sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of 53 KDa. Conclusions: Elevated activity of IMPDH was observed in S. aureus grown under anaerobic conditions and this was correlated with the biofilm formation indicating the linkage between purine metabolism and pathogenesis.

  6. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  7. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J. (Cornell); (UMC)

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  8. Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase[OPEN

    Science.gov (United States)

    Niehaus, Thomas D.; Hasnain, Ghulam; Gidda, Satinder K.; Nguyen, Thuy N.D.; Anderson, Erin M.; Brown, Greg; Yakunin, Alexander F.; de Crécy-Lagard, Valérie; Gregory, Jesse F.

    2016-01-01

    To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470. Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms. PMID:27677881

  9. Cyclic Adenosine Monophosphate Accumulation and beta-Adrenergic Binding in Unweighted and Denervated Rat Soleus Muscle

    Science.gov (United States)

    Kirby, Christopher R.; Woodman, Christopher R.; Woolridge, Dale; Tischler, Marc E.

    1992-01-01

    Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a p-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular (IM) injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (B(sub max)) for iodo-125(-)-pindolol in particulate preparations of unweighted (420 x 10(exp -18) mol/mg muscle) than of control or denervated muscles (285 x 10(exp-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished CAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +'- 6%, P less than .001). These results show that insulin affects CAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

  10. Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

    Science.gov (United States)

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D

    2013-05-23

    Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition, and gastroenteritis and poses a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5'-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and >500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD(+). The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An X-ray crystal structure of a representative E·IMP·inhibitor complex is also presented. Overall, the secondary amine derivative 15a demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines.

  11. Changes of nitric oxide synthase and cyclic guanosine monophosphate in form deprivation myopia in guinea pigs

    Institute of Scientific and Technical Information of China (English)

    WU Jie; LIU Qiong; YANG Xiao; YANG Hui; WANG Xin-mei; ZENG Jun-wen

    2007-01-01

    Background The form deprivation(FD)reduces spatial contrasts and induces myopia. Nitric oxide and cyclic guanosine monophosphate(cGMP)are involved in visual signal transmission.This study investigated changes in nitric oxide synthase(NOS)activity and cGMP concentration in ocular tissues in acute and chronic form deprivation myopia.Methods Guinea pigs had one eye covered by translucent glass for 7,14 or 21 days.Untreated litter mates were used as controls.NOS activity and cGMP concentrations in the retinal,choroidal and scleral tissues of FD eyes and controleyes were analyzed by radioimmunoassay after various durations of FD.The expression of NOS subtypes was identified by immunohistochemistry.Results Myopia was successfully induced in FD eyes after 14 days.Compared with control groups,the retinal NOS activity and cGMP concentrations in the FD eyes significantly increased after 14 and 21 days while the retinal NOS activity in the FD eyes was transiently suppressed by 7 days of FD.The NOS activity and cGMP concentrations of choroid and sclera in the FD eyes were higher than in the control groups at 21 days.The three isoenzymes of nitric oxide synthase were detected in the ocular tissues of guinea pigs.Conclusions The NOS activity and cGMP concentrations were upregulated after chronic FD and the retinal NOS activity was transiently suppressed at acute FD.The function of elevated NOS activity may be mediated by cGMP.

  12. Perfluorooctyl Iodide Stimulates Steroidogenesis in H295R Cells via a Cyclic Adenosine Monophosphate Signaling Pathway.

    Science.gov (United States)

    Wang, Chang; Ruan, Ting; Liu, Jiyan; He, Bin; Zhou, Qunfang; Jiang, Guibin

    2015-05-18

    Perfluorinated iodine alkanes (PFIs) are used widely in the organic fluorine industry. Increased production of PFIs has caused environmental health concerns. To evaluate the potential endocrine-disrupting effect of PFIs, we investigated the effects of perfluorooctyl iodide (PFOI) on steroidogenesis in human adrenocortical carcinoma cells (H295R). Levels of aldosterone, cortisol, 17β-estradiol, and testosterone were measured in H295R culture medium upon treatment with perfluorooctanoic acid (PFOA) and PFIs. Expression of 10 steroidogenic genes (StAR, HMGR, CYP11A1, 3βHSD2, 17βHSD, CYP17, CYP21, CYP11B1, CYP11B2, and CYP19) was measured by real-time polymerase chain reaction. Levels of cyclic adenosine monophosphate (cAMP) and adenylate cyclase (AC) activity were measured to understand the underlying mechanism of steroidogenic perturbations. Levels of production of aldosterone, cortisol, and 17β-estradiol were elevated significantly, and the level of testosterone generation decreased upon treatment with 100 μM PFOI. Similar to the effect induced by forskolin (AC activator), expression of all 10 genes involved in the synthesis of steroid hormones was upregulated significantly upon exposure to 100 μM PFOI. PFOA had no effect on steroid hormone production or steroidogenic gene expression even though it is highly structurally similar with PFOI. Therefore, the terminal -CF2I group in PFOI could be a critical factor for mediation of steroidogenesis. PFOI increased AC activity and cAMP levels in H295R cells, which implied an underlying mechanism for the disturbance of steroidogenesis. These data suggest that PFOI may act as an AC activator, thereby stimulating steroidogenesis by activating a cAMP signaling pathway.

  13. The mRNA Expression Status of Dopamine Receptor D2, Dopamine Receptor D3 and DARPP-32 in T Lymphocytes of Patients with Early Psychosis

    Directory of Open Access Journals (Sweden)

    Yin Cui

    2015-11-01

    Full Text Available Peripheral blood lymphocytes are an attractive tool because there is accumulating evidence indicating that lymphocytes may be utilized as a biomarker in the field of psychiatric study as they could reveal the condition of cells distributed in the brain. Here, we measured the mRNA expression status of dopamine receptor D2 (DRD2, DRD3, and dopamine and cyclic adenosine 3′,5′-monophosphate regulated phosphoprotein-32 (DARPP-32 in T lymphocytes of patients with early psychosis by quantitative real-time polymerase chain reaction (q-PCR and explored the relationships between their mRNA levels and the psychopathological status of patients. The present study demonstrated that the mRNA expression levels of DRD3 in T lymphocytes were significantly different among controls, and in patients with psychotic disorder not otherwise specified (NOS and schizophrenia/schizophreniform disorder. However, no significant differences in mRNA expression levels of DRD2 and DARPP-32 were found among the three groups. We found a significant positive correlation between the DRD2 mRNA level and the score of the excited factor of the Positive and Negative Syndrome Scale (PANSS in patients with schizophrenia/schizophreniform disorder. These findings suggest that DRD3 mRNA levels may serve as a potential diagnostic biomarker differentiating patients with early psychosis from controls.

  14. Evidence for mRNA expression of vascular endothelial growth factor by X-ray irradiation in a lung squamous carcinoma cell line.

    Science.gov (United States)

    Ando, S; Nojima, K; Majima, H; Ishihara, H; Suzuki, M; Furusawa, Y; Yamaguchi, H; Koike, S; Ando, K; Yamauchi, M; Kuriyama, T

    1998-10-23

    Vascular endothelial growth factor (VEGF) is a multipotent cytokine which plays an important role in various angiogenic conditions as well as in some tumor behaviors. Here we examined the induction of VEGF mRNA by X-ray irradiation in a lung squamous cell carcinoma cell line (RERF-LC-AI). Irradiating the cells with 15 Gy X-rays significantly increased the mRNA expression up to 2.5-fold of control at a post-irradiation time of 16-24 h. The induction of VEGF mRNA by X-ray irradiation was completely blocked by treating cells with either genistein (Src tyrosine kinase inhibitor) or H7 (protein kinase C inhibitor). This suggests that the mechanism of induction might be concerned with the pathway which triggers Src tyrosine kinase of the cell surface and the protein kinase C pathway.

  15. Kinetin in familial dysautonomia carriers: implications for a new therapeutic strategy targeting mRNA splicing.

    Science.gov (United States)

    Gold-von Simson, Gabrielle; Goldberg, Judith D; Rolnitzky, Linda M; Mull, James; Leyne, Maire; Voustianiouk, Andrei; Slaugenhaupt, Susan A; Axelrod, Felicia B

    2009-03-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IkappaB kinase-associated protein gene (IKBKAP) that leads to partial skipping of exon 20 and tissue-specific reduction of IkappaB kinase-associated protein/elongator protein 1 (IKAP/ELP-1 protein). Kinetin increases IKBKAP mRNA and protein expression in FD cell lines. To determine whether oral kinetin alters IKBKAP splicing in vivo, we administered kinetin to 29 healthy carriers of the major FD mutation for 8 d. Adverse effects, kinetin, and IKBKAP mRNA levels were monitored. In the highest dosing cohorts (23.5 mg/kg/d), the target plasma kinetin level was achieved in 91% of subjects at 2 h. After 8 d, IKBKAP mRNA expression in leukocytes increased as kinetin levels increased. There is a linear association between log plasma kinetin level and corresponding log change from baseline in IKBKAP mRNA expression that allows estimation of IKBKAP mRNA levels because of kinetin ingestion. Adverse effects were transient and mild. This is the first report of in vivo IKBKAP splicing modification and strongly suggests kinetin's therapeutic potential in FD and perhaps in other splicing disorders. Furthermore, our findings support our hypothesis that treatments, which target a particular splicing mutation, can be successfully developed.

  16. Application Of Kinase Inhibitors For Anti-Aging Intervention.

    Science.gov (United States)

    Cano, Mercedes; Ayala, Antonio; Marotta, Francesco; Argüelles, Sandro

    2017-07-14

    Protein phosphorylation, mediated by protein kinases, has important physiological and pathological implications in our lives . Targeting kinase is one of the most interesting of the emerging topics in the pharmaceutical industry, especially since there is a focus on cancer therapy. Given that kinases may be involved in the aging process the focus will be on using the kinase inhibitor for anti-aging intervention to enhance healthspan and increase longevity. In this review , we will summarize: (i) the impact of the phosphoproteomic approach to elucidate molecular mechanisms of diseases; (ii) importance of the drug discovery approach for targeting kinases; (iii) the dysregulation of Janus kinase (JAK) / signal-transducing adapter molecules (STAT) and p70 ribosomal protein S6 kinase (S6Ks) pathway in aging and the age-related process; (iv) the epidemiological studies available in order to see whether a correlation between JAK/STAT and S6Ks mRNA expression levels exist in cancer and in patient outcome; (v) finally, we will show selected inhibitors of these kinases approved by the US Food and Drug Administration (FDA). Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Adenosine 3':5'-cyclic monophosphate in higher plants: Isolation and characterization of adenosine 3':5'-cyclic monophosphate from Kalanchoe and Agave.

    Science.gov (United States)

    Ashton, A R; Polya, G M

    1977-07-01

    1.3':5'-Cyclic AMP was extensively purified from Kalanchoe daigremontiana and Agave americana by neutral alumina and anion- and cation-exchange column chromatography. Inclusion of 3':5'-cyclic [8-3H]AMP from the point of tissue extraction permitted calculation of yields. The purification procedure removed contaminating material that was shown to interfere with the 3':5'-cyclic AMP estimation and characterization procedures. 2. The partially purified 3':5'-cyclic AMP was quantified by means of a radiochemical saturation assay using an ox heart 3':5'-cyclic AMP-binding protein and by an assay involving activation of a mammalian protein kinase. 3. The plant 3':5'-cyclic AMP co-migrated with 3':5'-cyclic [8-3H]AMP on cellulose chromatography, poly(ethyleneimine)-cellulose chromatography and silica-gel t.l.c. developed with several solvent systems. 4. The plant 3':5'-cyclic AMP was degraded by ox heart 3':5'-cyclic nucleotide phosphodiesterase at the same rates as authentic 3':5'-cyclic AMP. 1-Methyl-3-isobutylxanthine (1 mM), a specific inhibitor of the 3':5'-cyclic nucleotide phosphodieterase, completely inhibited such degradation. 5. The concentrations of 3':5'-cyclic AMP satisfying the above criteria in Kalanchoe and Agave were 2-6 and 1 pmol/g fresh wt. respectively. Possible bacterial contribution to these analyses was estimated to be less than 0.002pmol/g fresh wt. Evidence for the occurrence of 3':5'-cyclic AMP in plants is discussed.

  18. Elevated nitric oxide and 3',5' cyclic guanosine monophosphate levels in patients with alcoholic cirrhosis

    Institute of Scientific and Technical Information of China (English)

    C(i)ntia Siqueira; Miguel Carneiro de Moura; Ana J(u)lia Pedro; Paula Rocha

    2008-01-01

    AIM: To evaluate whether serum levels of nitric oxide (NO') and plasma levels of cyclic guanosine monophosphate (Cgmp) and total glutathione (GSH) are altered in patients with alcoholic cirrhosis and to examine their correlation with the severity of liver disease.METHODS: Twenty-six patients with alcoholic liver cirrhosis were studied. Serum levels of NO· and plasma levels of cGMP and GSH were measured in 7 patients with compensated alcoholic cirrhosis (Child-Pugh A) and 19 patients with advanced cirrhosis (Child-Pugh B and C).The model for end-stage liver disease (MELD) score was evaluated. Sixteen healthy volunteers served as controls.Liver enzymes and creatinine levels were also tested.RESULTS: NO· and cGMP levels were higher in patients with Child-Pugh B and C cirrhosis than in Child-Pugh A cirrhosis or controls (NO·: 21.70 ± 8.07 vs 11.70 ± 2.74; 21.70 ± 8.07 vs 7.26 ± 2.47 μmol/L, respectively;P < 0.001) and (cGMP: 20.12 ± 6.62 vs 10.14 ± 2.78;20.12 ± 6.62 vs 4.95 ± 1.21 pmol/L, respectively; P <0.001). Total glutathione levels were lower in patients with Child-Pugh B and C cirrhosis than in patients with Child-Pugh A cirrhosis or controls (16.04 ± 6.06 vs 23.01 ± 4.38 or 16.04 ± 6.06 vs 66.57 ± 26.23 μmol/L,respectively; P < 0.001). There was a significant correlation between NO· and cGMP levels in all patients with alcoholic cirrhosis. A significant negative correlation between reduced glutathione/glutathione disulfide and the MELD score was found in all cirrhotic patients. CONCLUSION: Our results suggest a role for oxidative stress in alcoholic liver cirrhosis, which is more significant in decompensated patients with higher levels of NO· and cGMP and lower GSH levels than in compensated and control patients. Altered mediator levels in decompensated patients may influence the hemodynamic changes in and progression of liver disease.

  19. Hypothermia induced by adenosine 5'-monophosphate attenuates early stage injury in an acute gouty arthritis rat model.

    Science.gov (United States)

    Miao, Zhimin; Guo, Weiting; Lu, Shulai; Lv, Wenshan; Li, Changgui; Wang, Yangang; Zhao, Shihua; Yan, Shengli; Tao, Zhenyin; Wang, Yunlong

    2013-08-01

    To investigate whether the hypothermia induced by Adenosine 5'-Monophosphate (5'-AMP) could attenuate early stage injury in a rat acute gouty arthritis model. Ankle joint injection with monosodium urate monohydrate crystals (MSU crystals) in hypothermia rat model which was induced by 5'-AMP and then observe whether hypothermia induced by 5'-AMP could be effectively inhibit the inflammation on acute gouty arthritis in rats. AMP-induced hypothermia has protective effects on our acute gouty arthritis, which was demonstrated by the following criteria: (1) a significant reduction in the ankle swelling (p gouty arthritis model.

  20. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A;

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  1. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    Science.gov (United States)

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.

  2. Cellular stress increases RGS2 mRNA and decreases RGS4 mRNA levels in SH-SY5Y cells.

    Science.gov (United States)

    Song, Ling; Jope, Richard S

    2006-07-24

    Modulation of the expression of regulator of G-protein signaling (RGS) proteins is a major mechanism used to modulate their actions. Besides control by second messengers, the expression of RGS proteins, particularly RGS2, can be regulated by cell stress. Because RGS2 and RGS4 expression can be regulated by the cell cycle, we examined if cell cycle signals are involved in their regulation following stress. Treatment of SH-SY5Y cells with camptothecin increased RGS2 mRNA and decreased RGS4 mRNA levels. This effect on RGS2 mRNA was blocked by the cyclin-dependent kinase-2 (cdk2) inhibitors roscovitine and purvalanol. Cell cycle arrest was further implicated in regulating RGS mRNA levels because geldanamycin, which causes cell cycle arrest by inhibiting the actions of heat shock protein 90, caused changes in the mRNA levels of RGS2 and RGS4 similar to, and additive with, the effects of camptothecin. Overall, these results indicate that cell cycle arrest regulates the expression of RGS2 and RGS4, and that the expression of these two RGS family members is oppositely regulated by stress that causes cell cycle arrest.

  3. Involvement of second messengers in the signaling pathway of vitellogenesis-inhibiting hormone and their effects on vitellogenin mRNA expression in the whiteleg shrimp, Litopenaeus vannamei.

    Science.gov (United States)

    Bae, Sun-Hye; Okutsu, Tomoyuki; Tsutsui, Naoaki; Kang, Bong Jung; Chen, Hsiang-Yin; Wilder, Marcy N

    2017-05-15

    We incubated fragments of Litopenaeus vannamei ovary to investigate second messengers involved in the regulation of vitellogenin (vg) mRNA levels. The use of 100nM recombinant vitellogenesis-inhibiting hormone (VIH) (corresponding to recombinant L. vannamei sinus gland peptide-G: rLiv-SGP-G) significantly reduced vg mRNA expression in sub-adults after 8h incubation to less than 20% of the control. The concentration of intracellular cyclic guanosine monophosphate (cGMP) increased 3.2-fold relative to the control after 2h incubation with rLiv-SGP-G. However, it reached levels 18-fold relative to the control after 0.5h incubation with rLiv-SGP-G where 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) was also added. Moreover, vg mRNA expression was significantly reduced to less than 50% of the control after 24h incubation with 1μM A23187 (a calcium ionophore). Thus, rLiv-SGP-G and calcium ionophore reduced vg mRNA expression in in vitro-cultured ovary, and cGMP may be involved in the signaling pathway of VIH. Overall, the above results suggest that vg mRNA expression might be inhibited in vitro by increasing intracellular cGMP and Ca(2+) in L. vannamei ovary. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. A study of the hydration of deoxydinucleoside monophosphates containing thymine, uracil and its 5-halogen derivatives: Monte Carlo simulation.

    Science.gov (United States)

    Alderfer, J L; Danilov, V I; Poltev, V I; Slyusarchuk, O N

    1999-04-01

    An extensive Monte Carlo simulation of hydration of various conformations of the dinucleoside monophosphates (DNP), containing thymine, uracil and its 5-halogen derivatives has been performed. An anti-anti conformation is the most energetically stable one for each of the DNPs. In the majority of cases the energy preference is determined by water-water interaction. For other dimers conformational energy is the most important factor, or both the factors are of nearly equal importance. The introduction of the methyl group into the 5-position of uracil ring most noticeably influences the conformational energy and leads to the decrease of its stabilizing contribution to the total interaction energy. The introduction of halogen atoms increases the relative content of anti-syn and syn-anti conformations of DNPs as compared to the parent ones due to the formation of an energetically more favorable water structure around these conformations. A correlation is observed between the Monte Carlo results for the halogenated DNPs and their experimental photoproduct distribution. The data obtained demonstrates a sequence dependence in the photochemistry of the halogenated dinucleoside monophosphates.

  5. A novel procedure for purification of uridine 5'-monophosphate based on adsorption methodology using a hyper-cross-linked resin.

    Science.gov (United States)

    Wu, Jinglan; Zhu, Hui; Liu, Yanan; Zhou, Jingwei; Zhuang, Wei; Jiao, Pengfei; Ke, Xu; Ying, Hanjie

    2015-05-01

    The conventional ion exchange process used for recovery of uridine 5'-monophosphate (UMP) from the enzymatic hydrolysate of RNA is environmentally harmful and cost intensive. In this work, an innovative benign process, which comprises adsorption technology and use of a hyper-cross-linked resin as a stationary phase is proposed. The adsorption properties of this kind of resin in terms of adsorption equilibrium as well as kinetics were evaluated. The influences of the operating conditions, i.e., initial UMP concentration, feed flow rate, and bed height on the breakthrough curves of UMP in the fixed bed system were investigated. Subsequently, a chromatographic column model was established and validated for the prediction of the experimentally attained breakthrough curves of UMP and the main impurity component (phosphate ion) with a real enzymatic hydrolysate of RNA as a feed mixture. At the end of this paper, the crystallization of UMP was carried out. The purity of the final product (uridine 5'-monophosphate disodium, UMPNa2) of over 99.5 % was obtained.

  6. Correlation between indoleamine 2,3 dioxygenase mRNA and CDKN2A/p16 mRNA: a combined strategy to cervical cancer diagnosis.

    Science.gov (United States)

    Saffi Junior, Mario Cezar; Duarte, Ivone da Silva; Brito, Rodrigo Barbosa de Oliveira; Prado, Giovana Garcia; Makabe, Sergio; Dellê, Humberto; Camacho, Cleber P

    2016-11-01

    Cervical cancer (CC) is one of the most common cancers among women worldwide. The relation of the human papillomavirus (HPV) with CC and its precursor lesions was first suspected for over 40 years. The indoleamine 2,3 dioxygenase (IDO) is an immune modulator enzyme responsible for the immune system tissue protection mechanism, which may be the key to the tumoural persistence. HPV oncoprotein E7 promotes the increase in cyclin-dependent kinase inhibitor p16 (CDKN2A/p16). The isolated and combined analysis of CDKN2A/p16 mRNA to CC diagnosis was done with promising results. The aim of this study is to evaluate the correlation between IDO mRNA and CDKN2A/p16 mRNA. We will explore the potential of both as diagnostic tools. RNA was extracted from tissue samples. cDNA was generated with High Capacity RNA-to-cDNA kit. The real-time PCR results were analysed using nonlinear curve estimation, ROC curve, Chi-squared test, the proportion of variance explained and Galen and Gambino formulas. From 270 patients attended, colposcopy examination was performed in 110 and the biopsy in 75 patients. We found a positive correlation in patients older than 28 years old with low-risk lesions, but the correlation is lost in high-risk lesions. Although cytology, IDO mRNA and CDKN2A/p16 mRNA could not differentiate the risk groups, IDO combined with CDKN2A/p16 mRNA results could (p = 0.028). The best diagnostic result was achieved by IDO coupled with CDKN2A/p16 mRNA, which may considerably increase the sensitivity of screening for CC.

  7. Adenosine 5′-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation

    Science.gov (United States)

    Sun, Qi; Xu, Xi; Kong, Yi; Zhang, Jianfa

    2017-01-01

    Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the world. Hepatic c-jun NH2-terminal protein kinase (JNK) activation is thought to be a consequence of oxidative stress produced during APAP metabolism. Activation of JNK signals causes hepatocellular damage with necrotic and apoptotic cell death. Here we found that APAP caused a feedback increase in plasma adenosine 5′-monophsphate (5′-AMP). We demonstrated that co-administration of APAP and 5′-AMP significantly ameliorated APAP-induced hepatotoxicity in mice, without influences on APAP metabolism and its analgesic function. The mechanism of protection by 5′-AMP was through inhibiting APAP-induced activation of JNK, and attenuating downstream c-jun and c-fos gene expression. This was triggered by attenuating apoptosis signal-regulated kinase 1(ASK1) methylation and increasing ubiquitination-mediated ASK1 protein degradation. Our findings indicate that replacing the current APAP with a safe and functional APAP/5′-AMP formulation could prevent APAP-induced hepatotoxicity. PMID:28031524

  8. Reference: MRNA3ENDTAH3 [PLACE

    Lifescience Database Archive (English)

    Full Text Available MRNA3ENDTAH3 Ohtsubo N, Iwabuchi M The conserved 3'-flanking sequence, AATGGAAATG, ...of the wheat histone H3 gene is necessary for the accurate 3'-end formation of mRNA. Nucleic Acids Res 22:1052-1058 (1994) PubMed: 8152910; ...

  9. Design of Thymidine Analogues Targeting Thymidilate Kinase of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Luc Calvin Owono Owono

    2013-01-01

    Full Text Available We design here new nanomolar antituberculotics, inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt, by means of structure-based molecular design. 3D models of TMPKmt-inhibitor complexes have been prepared from the crystal structure of TMPKmt cocrystallized with the natural substrate deoxythymidine monophosphate (dTMP (1GSI for a training set of 15 thymidine analogues (TMDs with known activity to prepare a QSAR model of interaction establishing a correlation between the free energy of complexation and the biological activity. Subsequent validation of the predictability of the model has been performed with a 3D QSAR pharmacophore generation. The structural information derived from the model served to design new subnanomolar thymidine analogues. From molecular modeling investigations, the agreement between free energy of complexation (ΔΔGcom and Ki values explains 94% of the TMPKmt inhibition (pKi=-0.2924ΔΔGcom+3.234;R2=0.94 by variation of the computed ΔΔGcom and 92% for the pharmacophore (PH4 model (pKi=1.0206×pKipred-0.0832,  R2=0.92. The analysis of contributions from active site residues suggested substitution at the 5-position of pyrimidine ring and various groups at the 5′-position of the ribose. The best inhibitor reached a predicted Ki of 0.155 nM. The computational approach through the combined use of molecular modeling and PH4 pharmacophore is helpful in targeted drug design, providing valuable information for the synthesis and prediction of activity of novel antituberculotic agents.

  10. Myc Regulation of mRNA Cap Methylation

    Science.gov (United States)

    Cowling, Victoria H.; Cole, Michael D.

    2010-01-01

    The c-myc proto-oncogene regulates the expression of 15% to 20% of all genes, depending on the cell type, and the regulation is usually modest (1.5- to 2.0-fold). The authors discovered that in addition to regulating mRNA abundance, c-Myc regulates the formation of the 7-methylguanosine cap on many mRNAs, including transcriptional target genes and others not transcriptionally activated. Because the 7-methylguanosine cap is required for effective translation, enhanced methyl cap formation leads to increased protein production from Myc-responsive genes that exceeds the transcriptional induction. Increased cap methylation is linked to Myc-dependent enhanced activity of 2 critical kinases, TFIIH and p-TEFb, which phosphorylate the RNA polymerase II carboxy-terminal domain (CTD). Phosphorylation of the CTD recruits RNGTT and RNMT, the enzymes involved in mRNA capping, to the nascent transcript. Evidence is accumulating that enhanced cap methylation makes a significant contribution to Myc-dependent gene regulation and protein production. PMID:21170289

  11. Systems perspectives on mRNA processing

    Institute of Scientific and Technical Information of China (English)

    Adrienne E McKee; Pamela A Silver

    2007-01-01

    The application of genomic technologies to the study of mRNA processing is increasingly conducted in metazoan organisms in order to understand the complex events that occur during and after transcription. Large-scale systems analyses of mRNA-protein interactions and mRNA dynamics have revealed specificity in mRNA transcription, splicing, transport, translation, and turnover, and have begun to make connections between the different layers of mRNA processing. Here, we review global studies of post-transcriptional processes and discuss the challenges facing our understanding of mRNA regulation in metazoan organisms. In parallel, we examine genome-scale investigations that have expanded our knowledge of RNA-binding proteins and the networks of mRNAs that they regulate.

  12. Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

    Science.gov (United States)

    Soty, Maud; Chilloux, Julien; Delalande, François; Zitoun, Carine; Bertile, Fabrice; Mithieux, Gilles; Gautier-Stein, Amandine

    2016-04-01

    The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP.

  13. Compartmentalization Role of A-Kinase Anchoring Proteins (AKAPs in Mediating Protein Kinase A (PKA Signaling and Cardiomyocyte Hypertrophy

    Directory of Open Access Journals (Sweden)

    Abeer Rababa'h

    2014-12-01

    Full Text Available The Beta-adrenergic receptors (β-ARs stimulation enhances contractility through protein kinase-A (PKA substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs. AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα, phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2. Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure–function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.

  14. Mechanisms of mRNA translation of interferon stimulated genes.

    Science.gov (United States)

    Joshi, Sonali; Kaur, Surinder; Kroczynska, Barbara; Platanias, Leonidas C

    2010-01-01

    Over the last two decades, a lot of research work has been focused on the interferon (IFN)-regulated JAK-STAT pathway and understanding the mechanisms governing the transcription of interferon stimulated genes (ISGs). Evidence suggests that the JAK-STAT pathway alone does not account in its entirety for mediating cellular responses to IFNs. There is emerging evidence that non-Stat pathways play important roles in mediating signals for the generation of IFN-responses. Various studies have underscored the importance of mitogen activated protein kinases (MAPKs), especially p38 and ERK1/2, as well as the PI 3'K/AKT pathway in transmitting signals that are of critical importance for the biological effects of IFNs. Besides regulating the transcription of ISGs in some cases, engagement of these signaling pathways by the IFN-receptor (IFNR) associated complexes also plays an important role in mediating the translation of ISGs. The mechanisms regulating mRNA translation of ISGs is an area of ongoing active research and a lot more efforts will be required to complete our understanding of the various cellular elements involved in this process. In this review we highlight the mechanisms regulating translation of ISGs. We focus on the proteins regulated by the PI 3'K/AKT pathway, their role in mediating mRNA translation of ISGs and the functional consequences of this regulation. In addition, MAPKs are known to regulate the phosphorylation of various eukaryotic initiation factors and we summarize the roles of eIF4B and eIF4E phosphorylations on the translation of ISGs. The emerging roles of microRNAs in mRNA translation of ISGs are also discussed.

  15. Serine/Threonine Kinase 3-Phosphoinositide-Dependent Protein Kinase-1 (PDK1 as a Key Regulator of Cell Migration and Cancer Dissemination

    Directory of Open Access Journals (Sweden)

    Laura Di Blasio

    2017-03-01

    Full Text Available Dissecting the cellular signaling that governs the motility of eukaryotic cells is one of the fundamental tasks of modern cell biology, not only because of the large number of physiological processes in which cell migration is crucial, but even more so because of the pathological ones, in particular tumor invasion and metastasis. Cell migration requires the coordination of at least four major processes: polarization of intracellular signaling, regulation of the actin cytoskeleton and membrane extension, focal adhesion and integrin signaling and contractile forces generation and rear retraction. Among the molecular components involved in the regulation of locomotion, the phosphatidylinositol-3-kinase (PI3K pathway has been shown to exert fundamental role. A pivotal node of such pathway is represented by the serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1. PDK1, and the majority of its substrates, belong to the AGC family of kinases (related to cAMP-dependent protein kinase 1, cyclic Guanosine monophosphate-dependent protein kinase and protein kinase C, and control a plethora of cellular processes, downstream either to PI3K or to other pathways, such as RAS GTPase-MAPK (mitogen-activated protein kinase. Interestingly, PDK1 has been demonstrated to be crucial for the regulation of each step of cell migration, by activating several proteins such as protein kinase B/Akt (PKB/Akt, myotonic dystrophy-related CDC42-binding kinases alpha (MRCKα, Rho associated coiled-coil containing protein kinase 1 (ROCK1, phospholipase C gamma 1 (PLCγ1 and β3 integrin. Moreover, PDK1 regulates cancer cell invasion as well, thus representing a possible target to prevent cancer metastasis in human patients. The aim of this review is to summarize the various mechanisms by which PDK1 controls the cell migration process, from cell polarization to actin cytoskeleton and focal adhesion regulation, and finally, to discuss the evidence

  16. ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

    Science.gov (United States)

    Adachi, Shungo; Homoto, Masae; Tanaka, Rikou; Hioki, Yusaku; Murakami, Hiroshi; Suga, Hiroaki; Matsumoto, Masaki; Nakayama, Keiichi I; Hatta, Tomohisa; Iemura, Shun-ichiro; Natsume, Tohru

    2014-09-01

    Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.

  17. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

    Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics

  18. Mechanism of prostaglandin (PG)E2-induced prolactin expression in human T cells: cooperation of two PGE2 receptor subtypes, E-prostanoid (EP) 3 and EP4, via calcium- and cyclic adenosine 5'-monophosphate-mediated signaling pathways.

    Science.gov (United States)

    Gerlo, Sarah; Verdood, Peggy; Gellersen, Birgit; Hooghe-Peters, Elisabeth L; Kooijman, Ron

    2004-11-15

    We previously reported that prolactin gene expression in the T-leukemic cell line Jurkat is stimulated by PGE(2) and that cAMP acts synergistically with Ca(2+) or protein kinase C on the activation of the upstream prolactin promoter. Using the transcription inhibitor actinomycin D, we now show that PGE(2)-induced prolactin expression requires de novo prolactin mRNA synthesis and that PGE(2) does not influence prolactin mRNA stability. Furthermore, PGE(2)-induced prolactin expression was inhibited by protein kinase inhibitor fragment 14-22 and BAPTA-AM, which respectively, inhibit protein kinase A- and Ca(2+)-mediated signaling cascades. Using specific PGE(2) receptor agonists and antagonists, we show that PGE(2) induces prolactin expression through engagement of E-prostanoid (EP) 3 and EP4 receptors. We also found that PGE(2) induces an increase in intracellular cAMP concentration as well as intracellular calcium concentration via EP4 and EP3 receptors, respectively. In transient transfections, 3000 bp flanking the leukocyte prolactin promoter conferred a weak induction of the luciferase reporter gene by PGE(2) and cAMP, whereas cAMP in synergy with ionomycin strongly activated the promoter. Mutation of a C/EBP responsive element at -214 partially abolished the response of the leukocyte prolactin promoter to PGE(2), cAMP, and ionomycin plus cAMP.

  19. Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP–PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides

    Science.gov (United States)

    The cyclic AMP (cAMP)-PKA pathway is a central signaling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signaling in fungal biology has been well documented. Two key conserved components, adenylate cyclase (AC) and ca...

  20. Adenosine monophosphate forms ordered arrays in multilamellar lipid matrices: insights into assembly of nucleic acid for primitive life.

    Directory of Open Access Journals (Sweden)

    Laura Toppozini

    Full Text Available A fundamental question of biology is how nucleic acids first assembled and then were incorporated into the earliest forms of cellular life 4 billion years ago. The polymerization of nucleotides is a condensation reaction in which phosphodiester bonds are formed. This reaction cannot occur in aqueous solutions, but guided polymerization in an anhydrous lipid environment could promote a non-enzymatic condensation reaction in which oligomers of single stranded nucleic acids are synthesized. We used X-ray scattering to investigate 5'-adenosine monophosphate (AMP molecules captured in a multilamellar phospholipid matrix composed of dimyristoylphosphatidylcholine. Bragg peaks corresponding to the lateral organization of the confined AMP molecules were observed. Instead of forming a random array, the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity. This structure may facilitate polymerization of the nucleotides into RNA-like polymers.

  1. Brain-natriuretic peptide and cyclic guanosine monophosphate as biomarkers of myxomatous mitral valve disease in dogs

    DEFF Research Database (Denmark)

    Moesgaard, Sophia Gry; Falk, Bo Torkel; Teerlink, Tom

    2011-01-01

    Elevations in the plasma concentrations of natriuretic peptides correlate with increased severity of myxomatous mitral valve disease (MMVD) in dogs. This study correlates the severity of MMVD with the plasma concentrations of the biomarkers N-terminal fragment of the pro-brain-natriuretic peptide...... (NT-proBNP) and its second messenger, cyclic guanosine monophosphate (cGMP). Furthermore, the l-arginine:asymmetric dimethylarginine (ADMA) ratio was measured as an index of nitric oxide availability. The study included 75 dogs sub-divided into five groups based on severity of MMVD as assessed...... by clinical examination and echocardiography. Plasma NT-proBNP and cGMP concentrations increased with increasing valve dysfunction and were significantly elevated in dogs with heart failure. The cGMP:NT-proBNP ratio decreased significantly in dogs with heart failure, suggesting the development of natriuretic...

  2. Effects of Adenosine Monophosphate Used in Combination with L‐Arginine on Female Rabbit Corpus Cavernosum Tissue

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    Olivier Stücker, PhD

    2014-04-01

    Conclusions: Our results demonstrate that AMP induces a relaxing effect on the female rabbit corpora. They also show that L‐Arginine and AMP can potentiate each other and that a synergistic effect can be obtained by their combined use. Because only slight differences exist between both sexes in response to NO donors and/or nucleotide purines or in their use together, it is very likely that close biochemical mechanisms, although not to the same degree and not quite similar, are involved in the engorgement of the penis and the clitoris of New Zealand White rabbits. Stücker O, Pons C, Neuzillet Y, Laemmel E, and Lebret T. Original research‐sexual medicine: Effects of adenosine monophosphate used in combination with L‐Arginine on female rabbit corpus cavernosum tissue. Sex Med 2014;2:1–7.

  3. Crystallization and preliminary X-ray crystallographic analysis of adenosine 5′-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5′-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Han, Byung Woo [Department of Biochemistry, University of Wisconsin-Madison, WI 53706-1544 (United States); Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States); Bingman, Craig A. [Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States); Mahnke, Donna K.; Sabina, Richard L. [Department of Biochemistry, The Medical College of Wisconsin, Milwaukee, WI 53226-4801 (United States); Phillips, George N. Jr, E-mail: phillips@biochem.wisc.edu [Department of Biochemistry, University of Wisconsin-Madison, WI 53706-1544 (United States); Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States)

    2005-08-01

    Adenosine 5′-monophosphate deaminase from A. thaliana has been crystallized in complex with coformycin 5′-phosphate. Diffraction data have been collected to 3.34 Å resolution. Adenosine 5′-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5′-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5′-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6{sub 2}22, with unit-cell parameters a = b = 131.325, c = 208.254 Å, α = β = 90, γ = 120°. Diffraction data were collected to 3.34 Å resolution from a crystal in complex with coformycin 5′-phosphate and to 4.05 Å resolution from a crystal of a mercury derivative.

  4. Regulation and function of transaldolase isoenzymes involved in sugar and one-carbon metabolism in the ribulose monophosphate cycle methylotroph Arthrobacter P1

    NARCIS (Netherlands)

    Levering, P.R.; Dijkhuizen, Lubbert

    1986-01-01

    In the facultative methylotroph Arthrobacter P1 the enzyme transaldolase plays an important role in both the pentose phosphate pathway and in the ribulose monophosphate cycle of formaldehyde fixation. Among gluconate-negative mutants of Arthrobacter P1 strains occurred which also were unable to grow

  5. Magnetic and electronic transport properties of the monophosphate tungsten bronze (PO 2) 4(WO 3) 2 m, m = 2

    Science.gov (United States)

    Teweldemedhin, Z. S.; Ramanujachary, K. V.; Greenblatt, M.

    1991-11-01

    Large plate-like dark-brown crystals of monophosphate tungsten bronze (PO 2) 4(WO 3) 2 m, m = 2 or PWO 5 were prepared by reacting stoichiometric mixtures of P 2O 5, WO 3, and W at 1200°C. The temperature dependence of electrical resistivity along each of the three unique crystallographic axes of a single crystal shows semiconducting behavior down to 50 K with an activation energy of ˜0.084 eV. The room temperature resistivitity along the direction of corner sharing WO 6 octahedra is 5 × 10 -3 Ω · cm and about one to two orders of magnitude lower than along other unique directions, which implies quasi one-dimensional behavior. The magnetization study made on a batch of crystals in the temperature range of 2 to 300 K is indicative of antiferromagnetic ordering with a maximum at 15 K. An earlier theoretical study on the band electronic structure of (PO 2) 4(WO 3) 4 predicted both localized and delocalized electrons in narrow and dispersive bands, respectively. The observed magnetic moment of PWO 5 is consistent with the theoretical prediction, but the observed semiconductivity behavior is not. The difference in the observed electronic transport properties of PWO 5 from that of theoretically predicted behavior, as well as the anomalous magnetic and transport properties compared to the higher members of the series of the monophosphate tungsten bronzes {(PO 2) 4(WO 3) 2 m, m = 4, 6}, is discussed in terms of the unique structure of PWO 5.

  6. 5´AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Lundsgaard, Annemarie; Jeppesen, Jacob

    2015-01-01

    in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 -deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators, HDAC4 and SIRT1. Interestingly, PDK4 protein content...

  7. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  8. Enterococcus faecalis phosphomevalonate kinase.

    Science.gov (United States)

    Doun, Stephanie S; Burgner, John W; Briggs, Scott D; Rodwell, Victor W

    2005-05-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

  9. Rho-kinase inhibition ameliorates metabolic disorders through activation of AMPK pathway in mice.

    Directory of Open Access Journals (Sweden)

    Kazuki Noda

    Full Text Available BACKGROUND: Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK, a key molecule of metabolic conditions. METHODS AND RESULTS: Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O2 consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O2 consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1, with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C. CONCLUSIONS: These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that

  10. Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

    Science.gov (United States)

    Alonso, G D; Pereira, C A; Remedi, M S; Paveto, M C; Cochella, L; Ivaldi, M S; Gerez de Burgos, N M; Torres, H N; Flawiá, M M

    2001-06-01

    In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

  11. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

    Lee, Y.; Munnik, T.; Lee, Y.; Munnik, T.

    2010-01-01

    Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position of phosphoinositides. In Arabidopsis, only one PI3K exists, which belongs to the class-III PI3K subfamily which makes phosphatidylinositol 3-phosphate (PtdIns3P). The single AtPI3K gene is essential for survival, since loss of its

  12. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

    Lee, Y.; Munnik, T.; Munnik, T.

    2010-01-01

    Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position of phosphoinositides. In Arabidopsis, only one PI3K exists, which belongs to the class-III PI3K subfamily which makes phosphatidylinositol 3-phosphate (PtdIns3P). The single AtPI3K gene is essential for survival, since loss of its

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

    Science.gov (United States)

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

    2015-05-29

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

  14. Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures.

    Science.gov (United States)

    Schröder, H C; Diehl-Seifert, B; Rottmann, M; Messer, R; Bryson, B A; Agutter, P S; Müller, W E

    1988-03-01

    Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.

  15. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Oncogenic kinase NPM/ALK induces expression of HIF1a mRNA

    DEFF Research Database (Denmark)

    Marzec, M; Liu, X; Wong, W;

    2011-01-01

    to the HIF1a gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1a gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1a increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation...

  17. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress.

    Science.gov (United States)

    Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien; Lewis, Tommy L; Losón, Oliver C; Hellberg, Kristina; Young, Nathan P; Chen, Hsiuchen; Polleux, Franck; Chan, David C; Shaw, Reuben J

    2016-01-15

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission.

  18. Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress

    Science.gov (United States)

    Holmes, Brent; Artinian, Nicholas; Anderson, Lauren; Martin, Jheralyn; Masri, Janine; Cloninger, Cheri; Bernath, Andrew; Bashir, Tariq; Benavides-Serrato, Angelica; Gera, Joseph

    2011-01-01

    The A/U-rich RNA binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP regulated mRNA stability to mTORC2 activity. PMID:21964062

  19. Expression of PIK3CA, PTEN mRNA and PIK3CA mutations in primary breast cancer

    DEFF Research Database (Denmark)

    Palimaru, Irina; Brügmann, Anja; Wium-Andersen, Marie Kim;

    2013-01-01

    tissue samples of breast carcinoma and normal breast tissue were obtained from 175 breast cancer patients at the time of primary surgery, of these 105 patients were lymph node positive. Expression of PIK3CA and PTEN mRNA was quantified with Quantitative Real Time PCR. Somatic mutations in exon 9 and exon......PURPOSE: High activity of the intracellular phosphatidylinositol-3 kinase (PI3K) pathway is common in breast cancer. Here, we explore differences in expression of important PI3K pathway regulators: the activator, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA), and the tumour...... suppressor, phosphatase and tensin homolog (PTEN), in breast carcinoma tissue and normal breast tissue. Furthermore, we examine whether expression of PIK3CA and PTEN mRNA and occurrence of PIK3CA mutations are associated with lymph node metastases in patients with primary breast cancer. METHODS: Paired...

  20. Lack of cross-resistance to FF-10501, an inhibitor of inosine-5'-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell lines.

    Science.gov (United States)

    Murase, Motohiko; Iwamura, Hiroyuki; Komatsu, Kensuke; Saito, Motoki; Maekawa, Toshihiko; Nakamura, Takaaki; Yokokawa, Takuya; Shimada, Yasuhiro

    2016-02-01

    Resistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine-resistant cells derived from SKM-1 and MOLM-13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth-inhibitory effect of azacitidine, expression levels of uridine-cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross-resistance of greater than 10-fold (shift in IC50 value) was observed in azacitidine-resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors FF-10501 and mycophenolate mofetil (cross-resistance to 5-fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth-inhibitory activities in the azacitidine-resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF-10501 to its active form, FF-10501 ribosylmonophosphate [FF-10501RMP]), FF-10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF-10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.

  1. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia.

    Science.gov (United States)

    Axelrod, Felicia B; Liebes, Leonard; Gold-Von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A

    2011-11-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex-associated protein/elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase WT IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine whether oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/d for 28 d. An increase in WT IKBKAP mRNA expression in leukocytes was noted after 8 d in six of eight individuals; after 28 d, the mean increase compared with baseline was significant (p = 0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients but also that effect seems to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine whether kinetin will prove therapeutic in FD patients.

  2. CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

    Directory of Open Access Journals (Sweden)

    Eira Valeria Barrón

    Full Text Available The cyclin-dependent kinase inhibitor 3 (CDKN3 gene, involved in mitosis, is upregulated in cervical cancer (CC. We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa. CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney. A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17 died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression. In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

  3. CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

    Science.gov (United States)

    Barrón, Eira Valeria; Roman-Bassaure, Edgar; Sánchez-Sandoval, Ana Laura; Espinosa, Ana María; Guardado-Estrada, Mariano; Medina, Ingrid; Juárez, Eligia; Alfaro, Ana; Bermúdez, Miriam; Zamora, Rubén; García-Ruiz, Carlos; Gomora, Juan Carlos; Kofman, Susana; Pérez-Armendariz, E Martha; Berumen, Jaime

    2015-01-01

    The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

  4. Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

    Science.gov (United States)

    Deshmukh, Abhijit S.; Mitra, Pallabi; Maruthi, Mulaka

    2016-01-01

    Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5′-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis. PMID:27759017

  5. Evidence against mediation of adenosine-3',5'-cyclic monophosphate in the bud-inducing effect of cytokinins in moss protonemata

    Directory of Open Access Journals (Sweden)

    J. Scheneider

    2015-05-01

    Full Text Available Effects Oif adenosdne-3',5'-cyclic monophosphate (cAMP, N6,O2-dibuityryl adenosine-3',5'-cyclic monophosphate (DBcAMP, caffeine and theophylline on the bud-inducing activity of cytokinin in the protonema of two moss species, Ceratodon purpureus and Funaria hygrometrica were examined. The sub-stances have been found ineffective as gametophore bud inducers. Some synergism between cytokinin and cAMP or DBcAMP was observed with relation to the buds' growth, but this effect is nonspecific since it can be obtained with 5'-AMP or 5'-GMiP as well, The results seem to exclude the possibility of an involvement of cAMP as a second messenger in the mechanism of cytokinin action on morphogenetic processes in moss protonemata.

  6. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  7. Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis.

    Science.gov (United States)

    Sharma, Indra Mani; Prakash, Sunita; Dhanaraman, Thillaivillalan; Chatterji, Dipankar

    2014-10-01

    We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.

  8. Highly selective colorimetric detection of Ni2+ using silver nanoparticles cofunctionalized with adenosine monophosphate and sodium dodecyl sulfonate

    Science.gov (United States)

    Feng, Jiayu; Jin, Weiwei; Huang, Pengcheng; Wu, Fangying

    2017-09-01

    We report a dual-ligand strategy based on silver nanoparticles (AgNPs) for highly selective detection of Ni2+ using colorimetric techniques. Adenosine monophosphate (AMP) and sodium dodecyl sulfonate (SDS) were both used as ligands to modify AgNPs. The presence of Ni2+ induces the aggregation of AgNPs through cooperative electrostatic interaction and metal-ligand interaction, resulting in a color change from bright yellow to orange. The cofunctionalized AgNPs showed obvious advantages over the ones functionalized only by AMP or SDS in terms of selectivity. Under the optimized conditions, this sensing platform for Ni2+ works in the concentration range of 4.0 to 60 μM and has a low detection limit of 0.60 μM. In addition, the colorimetric assay is very fast, and the whole analysis can be completed within a few minutes. Thus, it can be directly used in tap water and lake water samples. [Figure not available: see fulltext.

  9. The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis.

    Science.gov (United States)

    Singh, Vinayak; Donini, Stefano; Pacitto, Angela; Sala, Claudia; Hartkoorn, Ruben C; Dhar, Neeraj; Keri, Gyorgy; Ascher, David B; Mondésert, Guillaume; Vocat, Anthony; Lupien, Andréanne; Sommer, Raphael; Vermet, Hélène; Lagrange, Sophie; Buechler, Joe; Warner, Digby F; McKinney, John D; Pato, Janos; Cole, Stewart T; Blundell, Tom L; Rizzi, Menico; Mizrahi, Valerie

    2017-01-13

    VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD(+). This compound binds at the NAD(+) site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

  10. Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host.

    Science.gov (United States)

    Santander, Javier; Mitra, Arindam; Curtiss, Roy

    2011-12-01

    Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.

  11. The expression of cyclic adenosine monophosphate responsive element modulator in rat sertoli cells following seminal extract administration

    Directory of Open Access Journals (Sweden)

    Muslim Akmal

    2016-09-01

    Full Text Available Aim: This study aims to determine the effect of seminal vesicle extract on cyclic adenosine monophosphate responsive element modulator (CREM expression in rat Sertoli cells. Materials and Methods: This study examined the expression of CREM on 20 male rats (Rattus norvegicus at 4 months of age, weighing 250-300 g. The rats were divided into four groups: K0, KP1, KP2, and KP3. K0 group was injected with 0.2 ml normal saline; KP1 was injected with 25 mg cloprostenol (Prostavet C, Virbac S. A; KP2 and KP3 were injected with 0.2 and 0.4 ml seminal vesicle extract, respectively. The treatments were conducted 5 times within 12-day interval. At the end of the study, the rats were euthanized by cervical dislocation; then, the testicles were necropsied and processed for histology observation using immunohistochemistry staining. Results: CREM expression in rat Sertoli cells was not altered by the administration of either 0.2 or 0.4 ml seminal vesicle extract. Conclusion: The administration of seminal vesicle extract is unable to increase CREM expression in rat Sertoli cells.

  12. Pharmacokinetic and pharmacodynamic analysis of inosine monophosphate dehydrogenase activity in hematopoietic cell transplantation recipients treated with mycophenolate mofetil.

    Science.gov (United States)

    Li, Hong; Mager, Donald E; Sandmaier, Brenda M; Storer, Barry E; Boeckh, Michael J; Bemer, Meagan J; Phillips, Brian R; Risler, Linda J; McCune, Jeannine S

    2014-08-01

    A novel approach to personalizing postgrafting immunosuppression in hematopoietic cell transplantation (HCT) recipients is evaluating inosine monophosphate dehydrogenase (IMPDH) activity as a drug-specific biomarker of mycophenolic acid (MPA)-induced immunosuppression. This prospective study evaluated total MPA, unbound MPA, and total MPA glucuronide plasma concentrations and IMPDH activity in peripheral blood mononuclear cells (PMNCs) at 5 time points after the morning dose of oral mycophenolate mofetil (MMF) on day +21 in 56 nonmyeloablative HCT recipients. Substantial interpatient variability in pharmacokinetics and pharmacodynamics was observed and accurately characterized by the population pharmacokinetic-dynamic model. IMPDH activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration in most patients. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory maximum effect model with an IC50 of 3.23 mg/L total MPA and 57.3 ng/mL unbound MPA. The day +21 IMPDH area under the effect curve (AUEC) was associated with cytomegalovirus reactivation, nonrelapse mortality, and overall mortality. In conclusion, a pharmacokinetic-dynamic model was developed that relates plasma MPA concentrations with PMNC IMPDH activity after an MMF dose in HCT recipients. Future studies should validate this model and confirm that day +21 IMPDH AUEC is a predictive biomarker.

  13. Barrier-free proton transfer in the valence anion of 2'-deoxyadenosine-5'-monophosphate. II. A computational study.

    Science.gov (United States)

    Kobyłecka, Monika; Gu, Jiande; Rak, Janusz; Leszczynski, Jerzy

    2008-01-28

    The propensity of four representative conformations of 2(')-deoxyadenosine-5(')-monophosphate (5(')-dAMPH) to bind an excess electron has been studied at the B3LYP6-31++G(d,p) level. While isolated canonical adenine does not support stable valence anions in the gas phase, all considered neutral conformations of 5(')-dAMPH form adiabatically stable anions. The type of an anionic 5(')-dAMPH state, i.e., the valence, dipole bound, or mixed (valence/dipole bound), depends on the internal hydrogen bond(s) pattern exhibited by a particular tautomer. The most stable anion results from an electron attachment to the neutral syn-south conformer. The formation of this anion is associated with a barrier-free proton transfer triggered by electron attachment and the internal rotation around the C4(')-C5(') bond. The adiabatic electron affinity of the a_south-syn anion is 1.19 eV, while its vertical detachment energy is 1.89 eV. Our results are compared with the photoelectron spectrum (PES) of 5(')-dAMPH(-) measured recently by Stokes et al., [J. Chem. Phys. 128, 044314 (2008)]. The computational VDE obtained for the most stable anionic structure matches well with the experimental electron binding energy region of maximum intensity. A further understanding of DNA damage might require experimental and computational studies on the systems in which purine nucleotides are engaged in hydrogen bonding.

  14. Simultaneous liquid chromatographic assessment of thiamine, thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics.

    Science.gov (United States)

    Mancinelli, Rosanna; Ceccanti, Mauro; Guiducci, Maria Soccorsa; Sasso, Guido Francesco; Sebastiani, Gemma; Attilia, Maria Luisa; Allen, John Paul

    2003-06-15

    An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (Pthiamine was established in the study of alcohol related problems.

  15. Convenient syntheses of 3'-amino-2',3'-dideoxynucleosides, their 5'-monophosphates, and 3'-aminoterminal oligodeoxynucleotide primers.

    Science.gov (United States)

    Eisenhuth, Ralf; Richert, Clemens

    2009-01-02

    5'-Protected 3'-amino-2',3'-dideoxynucleosides containing any of the four canonical nucleobases (A/C/G/T) were prepared via azides in five to six steps, starting from deoxynucleosides. For pyrimidines, the synthetic route involved nucleophilic opening of anhydronucleosides. For purines, an in situ oxidation/reduction sequence, followed by a Mitsunobu reaction with diphenyl-2-pyridylphosphine and sodium azide, provided the 3'-azidonucleosides in high yield and purity. For solid-phase synthesis of aminoterminal oligonucleotides, aminonucleosides were linked to controlled pore glass through a novel hexafluoroglutaric acid linker. These supports gave 3'-aminoterminal primers in high yield and purity via conventional DNA chain assembly and one-step deprotection/release with aqueous ammonia. Primers thus prepared were successfully tested in enzyme-free chemical primer extension, an inexpensive methodology for genotyping and labeling. Protected 5'-monophosphates of 3'-amino-2',3'-dideoxynucleosides were also prepared, providing starting materials for the preparation of labeled or photolably protected monomers for chemical primer extension.

  16. Inosine 5'-Monophosphate Dehydrogenase (IMPDH) as a Potential Target for the Development of a New Generation of Antiprotozoan Agents.

    Science.gov (United States)

    Fotie, Jean

    2016-06-19

    Inosine-5'-monophosphate dehydrogenase (IMPDH) is a metabolic enzyme that catalyzes the critical step in guanine nucleotide biosynthesis, and thus is at the center of cell growth and proliferation. However, although this enzyme has been exploited as potential target for the development of immunosuppressive, anticancer, and antiviral agents, the functional importance of IMPDH as a promising antiprotozoan drug target is still in its infancy mainly because of the availability of alternative nucleotides metabolic pathways in many of these parasites. This situation suggests that the inhibition of IMPDH might have little to no effect on the survival of protozoan parasites. As a result, no IMPDH inhibitor is currently commercially available or has advanced to clinical trials as a potential antiprotozoan drug. Nevertheless, recent advances toward the development of selective inhibitors of the IMPDH enzyme from Crystosporidium parvum as potential drug candidates against cryptosporidiosis should revive further investigations of this drug target in other protozoa parasites. The current review examines the chemical structures and biological activities of reported protozoan's IMPDH inhibitors. SciFinder was used to broadly pinpoint reports published on the topic in the chemical literature, with no specific time frame. Opportunities and challenges towards the development of inhibitors of IMPDH enzymes from protozoa parasites as potential chemotherapies toward the respective diseases they cause are also discussed.

  17. Determination of the electron-detachment energies of 2'-deoxyguanosine 5'-monophosphate anion: influence of the conformation.

    Science.gov (United States)

    Rubio, Mercedes; Roca-Sanjuán, Daniel; Serrano-Andrés, Luis; Merchán, Manuela

    2009-02-26

    The vertical electron-detachment energies (VDEs) of the singly charged 2'-deoxyguanosine 5'-monophosphate anion (dGMP-) are determined by using the multiconfigurational second-order perturbation CASPT2 method at the MP2 ground-state equilibrium geometry of relevant conformers. The origin of the unique low-energy band in the gas phase photoelectron spectrum of dGMP-, with maximum at around 5.05 eV, is unambiguously assigned to electron detachment from the highest occupied molecular orbital of pi-character belonging to guanine fragment of a syn conformation. The presence of a short H-bond linking the 2-amino and phosphate groups, the guanine moiety acting as proton donor, is precisely responsible for the pronounced decrease of the computed VDE with respect to that obtained in other conformations. As a whole, the present research supports the nucleobase as the site with the lowest ionization potential in negatively charged (deprotonated) nucleotides at the most stable conformations as well as for B-DNA-like type arrangements, in agreement with experimental evidence.

  18. The expression of cyclic adenosine monophosphate responsive element modulator in rat sertoli cells following seminal extract administration

    Science.gov (United States)

    Akmal, Muslim; Siregar, Tongku Nizwan; Wahyuni, Sri; Hamny; Nasution, Mustafa Kamal; Indriati, Wiwik; Panjaitan, Budianto; Aliza, Dwinna

    2016-01-01

    Aim: This study aims to determine the effect of seminal vesicle extract on cyclic adenosine monophosphate responsive element modulator (CREM) expression in rat Sertoli cells. Materials and Methods: This study examined the expression of CREM on 20 male rats (Rattus norvegicus) at 4 months of age, weighing 250-300 g. The rats were divided into four groups: K0, KP1, KP2, and KP3. K0 group was injected with 0.2 ml normal saline; KP1 was injected with 25 mg cloprostenol (Prostavet C, Virbac S. A); KP2 and KP3 were injected with 0.2 and 0.4 ml seminal vesicle extract, respectively. The treatments were conducted 5 times within 12-day interval. At the end of the study, the rats were euthanized by cervical dislocation; then, the testicles were necropsied and processed for histology observation using immunohistochemistry staining. Results: CREM expression in rat Sertoli cells was not altered by the administration of either 0.2 or 0.4 ml seminal vesicle extract. Conclusion: The administration of seminal vesicle extract is unable to increase CREM expression in rat Sertoli cells. PMID:27733803

  19. Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate

    Institute of Scientific and Technical Information of China (English)

    Jyun-Yi Wu; Chia-Hsin Chen; Li-Yin Yeh; Ming-Long Yeh; Chun-Chan Ting; Yan-Hsiung Wang

    2013-01-01

    Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J?cm22. Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J?cm22 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J?cm22 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.

  20. Isomerization mechanism of aspartate to isoaspartate implied by structures of Ustilago sphaerogena ribonuclease U2 complexed with adenosine 3'-monophosphate.

    Science.gov (United States)

    Noguchi, Shuji

    2010-07-01

    Aspartates in proteins are isomerized non-enzymatically to isoaspartate via succinimide in vitro and in vivo. In order to elucidate the mechanism of isoaspartate formation within the Asp45-Glu46 sequence of Ustilago sphaerogena ribonuclease U2 based on three-dimensional structure, crystal structures of ribonuclease U2 complexed with adenosine 3'-monophosphate have been solved at 0.96 and 0.99 A resolution. The crystal structures revealed that the C(gamma) atom of Asp45 is located just beside the main-chain N atom of Glu46 and that the conformation which is suitable for succinimide formation is stabilized by a hydrogen-bond network mediated by water molecules 190, 219 and 220. These water molecules are suggested to promote the formation of isoaspartate via succinimide: in the succinimide-formation reaction water 219 receives a proton from the N atom of Glu46 as a general base and waters 190 and 220 stabilize the tetrahedral intermediate, and in the succinimide-hydrolysis reaction water 219 provides a proton for the N atom of Glu46 as a general acid. The purine-base recognition scheme of ribonuclease U2 is also discussed.

  1. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  2. Enterococcus faecalis phosphomevalonate kinase

    OpenAIRE

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone D...

  3. Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system.

    Science.gov (United States)

    Schröder, H C; Rottmann, M; Bachmann, M; Müller, W E; McDonald, A R; Agutter, P S

    1986-08-15

    Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.

  4. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  5. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    Science.gov (United States)

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  6. Human taste and umami receptor responses to chemosensorica generated by Maillard-type N²-alkyl- and N²-arylthiomethylation of guanosine 5'-monophosphates.

    Science.gov (United States)

    Suess, Barbara; Brockhoff, Anne; Degenhardt, Andreas; Billmayer, Sylvia; Meyerhof, Wolfgang; Hofmann, Thomas

    2014-11-26

    Structural modification of the exocyclic amino function of guanosine 5'-monophosphate (5'-GMP) by Maillard-type reactions with reducing carbohydrates was recently found to increase the umami-enhancing activity of the nucleotide upon S-N(2)-1-carboxyalkylation and S-N(2)-(1-alkylamino)carbonylalkylation, respectively. Since the presence of sulfur atoms in synthetic N(2)-alkylated nucleotides was reported to be beneficial for sensory activity, a versatile Maillard-type modification of 5'-GMP upon reaction with glycine's Strecker aldehyde formaldehyde and organic thiols was performed in the present study. A series of N(2)-(alkylthiomethyl)guanosine and N(2)-(arylthiomethyl)guanosine 5'-monophosphates was generated and the compounds were evaluated to what extent they enhance the umami response to monosodium L-glutamate in vivo by a paired-choice comparison test using trained human volunteers and in vitro by means of cell-based umami taste receptor assay. Associated with a high umami-enhancing activity (β-value 5.1), N(2)-(propylthiomethyl)guanosine 5'-monophosphate could be generated when 5'-GMP reacted with glucose, glycine, and the onion-derived odorant 1-propanethiol, thus opening a valuable avenue to produce high-potency umami-enhancing chemosensorica from food-derived natural products by kitchen-type chemistry.

  7. Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

    Science.gov (United States)

    Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem

    2015-01-01

    Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

  8. NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  9. The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

    Science.gov (United States)

    Li, Ping; Stumpf, Maria; Müller, Rolf; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A

    2017-08-22

    SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

  10. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

    Institute of Scientific and Technical Information of China (English)

    Debby Ickowicz; Maya Finkelstein; Haim Breitbart

    2012-01-01

    Mammalian sperm must undergo a series of biochemical and physiological modifications,collectively called capacitation,in the female reproductive tract prior to the acrosome reaction (AR).The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2+ during both capacitation and the AR.In the present review,we summarize some of the signaling events that are involved in capacitation.During the capacitation process,phosphatidyl-inositol-3-kinase (P13K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade,and downregulated by protein kinase C α (PKCα).PKCα is active at the beginning of capacitation,resulting in P13K inactivation.During capacitation,PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism,allowing the activation of P13K.The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase.This activation of PKA leads to an increase in actin polymerization,an essential process for the development of hyperactivated motility,which is necessary for successful fertilization.Actin polymerization is mediated by PIP2 in two ways:first,P(I)P2 acts as a cofactor for phospholipase D (PLD) activation,and second,as a molecule that binds and inhibits actin-severing proteins such as gelsolin.Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation.Prior to the AR,gelsolin is released from P(I)P2 and undergoes dephosphorylation/activation,resulting in fast F-actin depolymerization,leading to the AR.

  11. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  12. Bmx regulates LPS-induced IL-6 and VEGF production via mRNA stability in rheumatoid synovial fibroblasts.

    Science.gov (United States)

    Palmer, Christine D; Mutch, Brenda E; Page, Theresa H; Horwood, Nicole J; Foxwell, Brian M J

    2008-06-13

    Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.

  13. Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP.

    Science.gov (United States)

    Serina, L; Blondin, C; Krin, E; Sismeiro, O; Danchin, A; Sakamoto, H; Gilles, A M; Bârzu, O

    1995-04-18

    The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  15. RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

    Science.gov (United States)

    Lee, Ye-Ji; Park, Hyun-Jung; Woo, So-Youn; Park, Eun-Mi; Kang, Jihee Lee

    2014-09-01

    Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

  16. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  17. Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking.

    Science.gov (United States)

    Dubey, Badri N; Lori, Christian; Ozaki, Shogo; Fucile, Geoffrey; Plaza-Menacho, Ivan; Jenal, Urs; Schirmer, Tilman

    2016-09-01

    Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di-guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling.

  18. Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking

    Science.gov (United States)

    Dubey, Badri N.; Lori, Christian; Ozaki, Shogo; Fucile, Geoffrey; Plaza-Menacho, Ivan; Jenal, Urs; Schirmer, Tilman

    2016-01-01

    Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di–guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling. PMID:27652341

  19. Multiple Decay Mechanisms and 2D‐UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine‐Uracil Monophosphate

    Science.gov (United States)

    Li, Quansong; Giussani, Angelo; Segarra‐Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A.; Mukamel, Shaul; Roca‐Sanjuán, Daniel

    2016-01-01

    Abstract The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D‐UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine 1La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine 1Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter‐base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long‐lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the 1Lb, S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D‐UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm−1 in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D‐UV spectroscopy to disentangle the photophysics of multichromophoric systems. PMID:27113273

  20. Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate.

    Science.gov (United States)

    Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís

    2016-05-23

    The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D-UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter-base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long-lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the (1) Lb , S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D-UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm(-1) in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D-UV spectroscopy to disentangle the photophysics of multichromophoric systems.

  1. Enhanced Molecular Recognition between Nucleobases and Guanine-5'-monophosphate-disodium (GMP) by Surfactant Aggregates in Aqueous Solution.

    Science.gov (United States)

    Liu, Zhang; Wang, Dong; Cao, Meiwen; Han, Yuchun; Xu, Hai; Wang, Yilin

    2015-07-15

    Only specific base pairs on DNA can bind with each other through hydrogen bonds, which is called the Watson-Crick (W/C) pairing rule. However, without the constraint of DNA chains, the nucleobases in bulk aqueous solution usually do not follow the W/C pairing rule anymore because of the strong competitive effect of water and the multi-interaction edges of nucleobases. The present work applied surfactant aggregates noncovalently functionalized by nucleotide to enhance the recognition between nucleobases without DNA chains in aqueous solution, and it revealed the effects of their self-assembling ability and morphologies on the recognition. The cationic ammonium monomeric, dimeric, and trimeric surfactants DTAB, 12-3-12, and 12-3-12-3-12 were chosen. The surfactants with guanine-5'-monophosphate-disodium (GMP) form micelles, vesicles, and fingerprint-like and plate-like aggregates bearing the hydrogen-bonding sites of GMP, respectively. The binding parameters of these aggregates with adenine (A), uracil (U), guanine (G), and cytosine(C) indicate that the surfactants can promote W/C recognitions in aqueous solution when they form vesicles (GMP/DTAB) or plate-like aggregates (GMP/12-3-12) with proper molecular packing compactness, which not only provide hydrophobic environments but also shield non-W/C recognition edges. However, the GMP/12-3-12 micelles with loose molecular packing, the GMP/12-3-12 fingerprint-like aggregates where the hydrogen bond sites of GMP are occupied by itself, and the GMP/12-3-12-3-12 vesicles with too strong self-assembling ability cannot promote W/C recognition. This work provides insight into how to design self-assemblies with the performance of enhanced molecule recognition.

  2. Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model.

    Science.gov (United States)

    Ross, Christina L; Teli, Thaleia; Harrison, Benjamin S

    2016-01-01

    During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

  3. Crystallographic study of Glu58Ala RNase T1 x 2'-guanosine monophosphate at 1.9-A resolution.

    Science.gov (United States)

    Pletinckx, J; Steyaert, J; Zegers, I; Choe, H W; Heinemann, U; Wyns, L

    1994-02-22

    Glu58 is known to participate in phosphodiester transesterification catalyzed by the enzyme RNase T1. For Glu58 RNase T1, an altered mechanism has been proposed in which His40 replaces Glu58 as the base catalyst [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. Glu58Ala Rnase T1 has been cocrystallized with guanosine 2'-monophosphate (2'-GMP). The crystals are of space group P2(1), with one molecule per asymmetric unit (a = 32.44 A, b = 49.64 A, c = 26.09 A, beta = 99.17 degrees). The three-dimensional structure of the enzyme was determined to a nominal resolution of 1.9 A, yielding a crystallographic R factor of 0.178 for all X-ray data. Comparison of this structure with wild-type structures leads to the following conclusions. The minor changes apparent in the tertiary structure can be explained by either the mutation of Glu58 or by the change in the space group. In the active site, the extra space available through the mutation of Glu58 is occupied by the phosphate group (after a reorientation) and by a solvent molecule replacing a carboxylate oxygen of Glu58. This solvent molecule is a candidate for participation in the altered mechanism of this mutant enzyme. Following up on a study of conserved water sites in RNase T1 crystallized in space group P2(1)2(1)2(1) [Malin, R., Zielenkiewicz, P., & Saenger, W. (1991) J. Mol. Biol. 266, 4848-4852], we investigated the hydration structure for four different packing modes of RNase T1.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Thiamine diphosphate in whole blood, thiamine and thiamine monophosphate in breast-milk in a refugee population.

    Directory of Open Access Journals (Sweden)

    Wolfgang Stuetz

    Full Text Available BACKGROUND: The provision of high doses of thiamine may prevent thiamine deficiency in the post-partum period of displaced persons. METHODOLOGY/PRINCIPAL FINDINGS: The study aimed to evaluate a supplementation regimen of thiamine mononitrate (100 mg daily at the antenatal clinics in Maela refugee camp. Women were enrolled during antenatal care and followed after delivery. Samples were collected at 12 weeks post partum. Thiamine diphosphate (TDP in whole blood and thiamine in breast-milk of 636 lactating women were measured. Thiamine in breast-milk consisted of thiamine monophosphate (TMP in addition to thiamine, with a mean TMP to total thiamine ratio of 63%. Mean whole blood TDP (130 nmol/L and total thiamine in breast-milk (755 nmol/L were within the upper range reported for well-nourished women. The prevalence of women with low whole blood TDP (<65 nmol/L was 5% and with deficient breast-milk total thiamine (<300 nmol/L was 4%. Whole blood TDP predicted both breast-milk thiamine and TMP (R(2 = 0.36 and 0.10, p<0.001. A ratio of TMP to total thiamine ≥63% was associated with a 7.5 and 4-fold higher risk of low whole blood TDP and deficient total breast-milk thiamine, respectively. Routine provision of daily 100 mg of thiamine mononitrate post-partum compared to the previous weekly 10 mg of thiamine hydrochloride resulted in significantly higher total thiamine in breast-milk. CONCLUSIONS/SIGNIFICANCE: Thiamine supplementation for lactating women in Maela refugee camp is effective and should be continued. TMP and its ratio to total thiamine in breast-milk, reported for the first time in this study, provided useful information on thiamine status and should be included in future studies of breast-milk thiamine.

  5. Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China); Lu, Weiqiang, E-mail: wqlu@bio.ecnu.edu.cn [Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Huang, Jin, E-mail: huangjin@ecust.edu.cn [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China)

    2016-09-02

    Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

  6. Amplified Peroxidase-Like Activity in Iron Oxide Nanoparticles Using Adenosine Monophosphate: Application to Urinary Protein Sensing.

    Science.gov (United States)

    Yang, Ya-Chun; Wang, Yen-Ting; Tseng, Wei-Lung

    2017-03-08

    Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe3O4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe3O4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H2O2) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe3O4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe(2+)/Fe(3+) sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H2O2. Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe3O4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe3O4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe3O4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe3O4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe3O4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

  7. Methacholine and adenosine 5'-monophosphate (AMP) responsiveness, and the presence and degree of atopy in children with asthma.

    Science.gov (United States)

    Suh, Dong I; Lee, Ju K; Kim, Chang K; Koh, Young Y

    2011-02-01

    The relationship between atopy and bronchial hyperresponsiveness (BHR), both key features of asthma, remains to be clarified. BHR is commonly evaluated by bronchial challenges using direct and indirect stimuli. The aim of this study was to investigate the degree of BHR to methacholine (direct stimulus) and adenosine 5'-monophosphate (AMP) (indirect stimulus) according to the presence and degree of atopy in children with asthma. We performed a retrospective analysis of data from 120 children presenting with a diagnosis of asthma. These children were characterized by skin-prick tests (SPTs), spirometry and bronchial challenges with methacholine and AMP. Atopy was defined by at least one positive reaction to SPTs, and its degree was measured using serum total IgE levels, number of positive SPTs and atopic scores (sum of graded wheal size). A provocative concentration causing a 20% decline in FEV(1) (PC(20) ) was determined for each challenge. Patients with atopy(n=94) had a significantly lower AMP PC(20) than non-atopic patients (n=26), whereas methacholine PC(20) was not different between the two groups. Among the patients with atopy, there was no association between methacholine PC(20) and any atopy parameter. In contrast, a significant association was found between AMP PC(20) and the degree of atopy reflected in serum total IgE, number of positive SPTs and atopic scores (anova trend test, p=0.002, 0.001, 0.003, respectively). AMP responsiveness was associated with the presence and degree of atopy, whereas such a relationship was not observed for methacholine responsiveness. These findings suggest that atopic status may be better reflected by bronchial responsiveness assessed by AMP than by methacholine.

  8. Phosphatidylinositol 3-kinase in myogenesis.

    Science.gov (United States)

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc.

  9. Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK

    DEFF Research Database (Denmark)

    Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik

    2016-01-01

    Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding ......Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element......-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory......-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet...

  10. Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

    Science.gov (United States)

    Gemba, Takefumi; Valbracht, Jean; Alsalameh, Saifeddin; Lotz, Martin

    2002-01-11

    The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.

  11. Sevoflurane effects on cyclic adenosine monophosphate response element binding protein, phosphorylated cyclic adenosine monophosphate response element binding protein, and Livin expression in the cortex and hippocampus of a vascular cognitive impairment rat model

    Institute of Scientific and Technical Information of China (English)

    Bin Wu; Ling Dan; Xianlin Zhu

    2009-01-01

    BACKGROUND: Neuronal necrosis and apoptosis play important roles in the pathophysiology of cerebral ischemia and resulting cognitive impairment. However, inhibition of neuronal necrosis and apoptosis has been shown to attenuate cognitive impairment following cerebral ischemia.OBJECTIVE: To investigate the effects of sevoflurane on cyclic adenosine monophosphate response element binding protein (CREB), phosphorylated CREB (pCREB), and Livin expression in the cortex and hippocampus of a rat model of vascular cognitive impairment.DESIGN, TIME AND SETTING: A randomized, controlled experiment was performed in the Chongqing Key Laboratory of Neurology between June 2007 and July 2008.MATERIALS: Sevoflurane was provided by Abbott Laboratory, UK; Morris water maze was provided by Chinese Academy of Medical Sciences, China; goat anti-rat CREB, goat anti-rat pCREB and goat anti-rat Livin antibodies were provided by Biosource International, USA.METHODS: A total of 42 female, Wistar rats were randomly assigned to the following groups: sham operation, vascular cognitive impairment, and sevoflurane treatment. The vascular cognitive impairment rat model was established by permanent bilateral occlusion of both common carotid arteries, and 1.0 MAC sevoflurane was immediately administered by inhalation for 2 hours.MAIN OUTCOME MEASURES: CREB, pCREB, and Livin expression was measured in the cortex and hippocampus by Western blot and reverse transcription-polymerase chain reaction. Behavior was evaluated with Morris water maze.RESULTS: CREB, pCREB, and Livin expression in the sevoflurane treatment group was significantly greater than the vascular cognitive impairment group (P<0.01). However, expression of CREB and pCREB was significantly less in the sevoflurane treatment and vascular cognitive impairment groups, compared with the sham operation group (P<0.01). Livin expression in the sevoflurane treatment and vascular cognitive impairment groups was significantly greater than the sham

  12. Blockades of mitogen-activated protein kinase and calcineurin both change fibre-type markers in skeletal muscle culture

    DEFF Research Database (Denmark)

    Higginson, James; Wackerhage, Henning; Woods, Niall

    2002-01-01

    A and mitogen-activated protein kinase kinase (MEK1/2) blockade with U0126 upon myosin heavy chain (MHC) isoform mRNA levels and activities of metabolic enzymes after 1 day, 3 days and 7 days of treatment in primary cultures of spontaneously twitching rat skeletal muscle. U0126 treatment significantly decreased......Activation of either the calcineurin or the extracellular signal-regulated kinase (ERK1/2) pathway increases the percentage of slow fibres in vivo suggesting that both pathways can regulate fibre phenotypes in skeletal muscle. We investigated the effect of calcineurin blockade with cyclosporin...

  13. Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway.

    Science.gov (United States)

    Miller, Aimee L; Suntharalingam, Mythili; Johnson, Sylvia L; Audhya, Anjon; Emr, Scott D; Wente, Susan R

    2004-12-03

    Production of inositol hexakisphosphate (IP6) by Ipk1, the inositol-1,3,4,5,6-pentakisphosphate 2-kinase, is required for Gle1-mediated mRNA export in Saccharomyces cerevisiae cells. To examine the network of interactions that require IP6 production, an analysis of fitness defects was conducted in mutants harboring both an ipk1 null allele and a mutant allele in genes encoding nucleoporins or transport factors. Enhanced lethality was observed with a specific subset of mutants, including nup42, nup116, nup159, dbp5, and gle2, all of which had been previously connected to Gle1 function. Complementation of the nup116Deltaipk1Delta and nup42Deltaipk1Delta double mutants did not require the Phe-Gly repeat domains in the respective nucleoporins, suggesting that IP6 was acting subsequent to heterogeneous nuclear ribonucleoprotein targeting to the nuclear pore complex. With Nup42 and Nup159 localized exclusively to the nuclear pore complex cytoplasmic side, we speculated that IP6 may regulate a cytoplasmic step in mRNA export. To test this prediction, the spatial requirements for the production of IP6 were investigated. Restriction of Ipk1 to the cytoplasm did not block IP6 production. Moreover, coincident sequestering of both Ipk1 and Mss4 (an enzyme required for phosphatidylinositol 4,5-bisphosphate production) to the cytoplasm also did not block IP6 production. Given that the kinase required for inositol 1,3,4,5,6-pentakisphosphate production (Ipk2) is localized in the nucleus, these results indicated that soluble inositides were diffusing between the nucleus and the cytoplasm. Additionally, the cytoplasmic production of IP6 by plasma membrane-anchored Ipk1 rescued a gle1-2 ipk1-4 synthetic lethal mutant. Thus, cytoplasmic IP6 production is sufficient for mediating the Gle1-mRNA export pathway.

  14. A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

    Science.gov (United States)

    Kim, Y S; Nosaka, K; Downs, D M; Kwak, J M; Park, D; Chung, I K; Nam, H G

    1998-08-01

    We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

  15. BDNF and trkB mRNA expression in the rat hippocampus following entorhinal cortex lesions.

    Science.gov (United States)

    Lapchak, P A; Araujo, D M; Hefti, F

    1993-02-01

    Quantitative in situ hybridization was used to determine whether the prevalence or topographical distribution of brain-derived neurotrophic factor (BDNF) or tyrosine receptor kinase (trk) B mRNA is altered in the hippocampal formation following lesions of excitatory afferents from the entorhinal cortex which provides an external source of innervation for the hippocampal formation. BDNF mRNA levels were not altered in the hippocampal formation up to 10 days following entorhinal cortex lesions (ECLs). The levels of mRNA coding for all known forms of trkB receptors also remained unchanged. The prevalence of the synaptic plasticity marker SNAP-25 mRNA was increased in the CA2 and CA3 pyramidal cell layers and the dentate gyrus by 6 days following ECLs and remained elevated at 10 days following ECLs. Our findings indicate that hippocampal neuron sprouting which occurs in response to ECLs is not the result of changes in the expression of the BDNF or trkB mRNA.

  16. Expression of cell cycle regulating factor mRNA in small cell lung cancer xenografts

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1998-01-01

    We have investigated the expression of cyclins, cyclin dependent kinases (CDK), and CDK inhibitors (CKI) at the mRNA level in a panel of small-cell lung cancer (SCLC) cell lines in vitro and in vivo as xenografts in nude mice. The results showed that the cell lines expressed varying amounts of most...... cyclin and CDK's but only a few of the cell lines expressed cyclin D1 and/or D2 and some lacked expression of CDK6. Most cell lines expressed mRNA for the CKI's but two cell lines lacked expression of P15INK4B and p16INK4A. The mRNA expression differed for a few of the cell lines regarding cyclin D2...... and CDK6 when in vitro and in vivo data were compared. Two of the cell lines that express the retinoblastoma (Rb) protein had no sign of a deregulated Rb pathway but further studies at the protein level are necessary to demonstrate whether these two cell lines should have a normal Rb pathway or whether...

  17. Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening.

    Science.gov (United States)

    Bazin, Jérémie; Langlade, Nicolas; Vincourt, Patrick; Arribat, Sandrine; Balzergue, Sandrine; El-Maarouf-Bouteau, Hayat; Bailly, Christophe

    2011-06-01

    After-ripening is the mechanism by which dormant seeds become nondormant during their dry storage after harvest. The absence of free water in mature seeds does not allow detectable metabolism; thus, the processes associated with dormancy release under these conditions are largely unknown. We show here that sunflower (Helianthus annuus) seed alleviation of dormancy during after-ripening is associated with mRNA oxidation and that this oxidation is prevented when seeds are maintained dormant. In vitro approaches demonstrate that mRNA oxidation results in artifacts in cDNA-amplified fragment length polymorphim analysis and alters protein translation. The oxidation of transcripts is not random but selective, and, using microarrays, we identified 24 stored mRNAs that became highly oxidized during after-ripening. Oxidized transcripts mainly correspond to genes involved in responses to stress and in cell signaling. Among them, protein phosphatase 2C PPH1, mitogen-activated protein kinase phosphatase 1, and phenyl ammonia lyase 1 were identified. We propose that targeted mRNA oxidation during dry after-ripening of dormant seeds could be a process that governs cell signaling toward germination in the early steps of seed imbibition.

  18. An uncharacterized member of the ribokinase family in Thermococcus kodakarensis exhibits myo-inositol kinase activity.

    Science.gov (United States)

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-07-19

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

  19. Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan

    Science.gov (United States)

    Floor, Stephen N.; Borja, Mark S.; Gross, John D.

    2012-01-01

    Conformational dynamics in bilobed enzymes can be used to regulate their activity. One such enzyme, the eukaryotic decapping enzyme Dcp2, controls the half-life of mRNA by cleaving the 5′ cap structure, which exposes a monophosphate that is efficiently degraded by exonucleases. Decapping by Dcp2 is thought to be controlled by an open-to-closed transition involving formation of a composite active site with two domains sandwiching substrate, but many details of this process are not understood. Here, using NMR spectroscopy and enzyme kinetics, we show that Trp43 of Schizosaccharomyces pombe Dcp2 is a conserved gatekeeper of this open-to-closed transition. We find that Dcp2 samples multiple conformations in solution on the millisecond-microsecond timescale. Mutation of the gatekeeper tryptophan abolishes the dynamic behavior of Dcp2 and attenuates coactivation by a yeast enhancer of decapping (Edc1). Our results determine the dynamics of the open-to-closed transition in Dcp2, suggest a structural pathway for coactivation, predict that Dcp1 directly contacts the catalytic domain of Dcp2, and show that coactivation of decapping by Dcp2 is linked to formation of the composite active site. PMID:22323607

  20. Mechanism of the Orotidine 5’-Monophosphate Decarboxylase-Catalyzed Reaction: Importance of Residues in the Orotate Binding Site†

    Science.gov (United States)

    Iiams, Vanessa; Desai, Bijoy J.; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Gerlt, John A.

    2011-01-01

    The reaction catalyzed by orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for the rate enhancement [kcat/knon = 7.1 × 1016] and catalytic proficiency [(kcat/KM)/knon = 4.8 × 1022 M−1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that “substrate destabilization” by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ~5 kcal/mol (contributing ~2.7 × 103 to the rate enhancement) [K. K. Chan, B. M. Wood, A. A. Fedorov, E. V. Fedorov, H. J. Imker, T. L. Amyes, J. P. Richard, S. C. Almo, and J. A. Gerlt (2009) Biochemistry 48, 5518–31]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of kcat by as much as 400-fold but have minimal effect on the value of kex for exchange of H6 of the FUMP product analog with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions for Ser 127 that is proximal to O4 of the orotate ring decrease the value of kcat/KM, with the S127P substitution that eliminates hydrogen-bonding interactions with O4 producing a 2.5 × 106-fold reduction in the value of kcat/KM; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 to produce an anionic carbene intermediate and thereby provide a structural strategy for stabilization of the intermediate. These observations provide additional information on the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the

  1. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    Science.gov (United States)

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  2. Application of multiplexed kinase inhibitor beads to study kinome adaptations in drug-resistant leukemia.

    Directory of Open Access Journals (Sweden)

    Matthew J Cooper

    Full Text Available Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML. However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs and quantitative mass spectrometry (MS to compare kinase expression and activity in an imatinib-resistant (MYL-R and -sensitive (MYL cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9 or decreased (Abl, Kit, JNK, ATM, Yes abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942. MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244. Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify

  3. A label-free and self-assembled electrochemical biosensor for highly sensitive detection of cyclic diguanylate monophosphate (c-di-GMP) based on RNA riboswitch.

    Science.gov (United States)

    Xie, Qingyun; Zhao, Fulin; Liu, Hongrui; Shan, Yanke; Liu, Fei

    2015-07-02

    Cyclic diguanylate monophosphate (c-di-GMP) is an important second messenger that regulates a variety of complex physiological processes involved in motility, virulence, biofilm formation and cell cycle progression in several bacteria. Herein we report a simple label-free and self-assembled RNA riboswitch-based biosensor for sensitive and selective detection of c-di-GMP. The detectable concentration range of c-di-GMP is from 50 nM to 1 μM with a detection limit of 50 nM.

  4. Expression of the Apoptosis Inhibitor Survivin and its correlation with Thymidine Kinase and Axillary Lymph Node Metastasis in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Jian-Ping WU; Yun-Feng ZHOU; Zhi-Guo LUO; Ming-Sheng ZHANG

    2005-01-01

    @@ 1 Introduction Many molecular factors have been demonstrated to interfere with cellular proliferation and programmed cell death. One of these factors is a recently discovered member of the "inhibitor of apoptosis protein(IAP)" called survivin. Survivin is abundantly expressed in most solid and hematologic malignancies, but undetectable in normal adult tissues. Interference with survivin function induces pleiotropic cell-division defects and apoptosis. Cytosolic thymidine kinase (TK) is a marker for proliferating cells and TK is one of several key enzymes involved in DNAmetabolism that phosphorylates thymidine to thymidine mono-phosphate. This study was aimed to detect the expression of suvivin and TK in breast cancer, and to explore a possible relationship between survivin expression and axillary lymph node metastasis.

  5. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  6. FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

    Science.gov (United States)

    Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

    2014-04-01

    Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

  7. Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

    Science.gov (United States)

    Kanai, Yukiko; Asano, Kosuke; Komatsu, Yoshiko; Takagi, Katsuhiro; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Haneishi, Ayumi; Tsukada, Akiko; Yamada, Kazuya

    2017-02-01

    The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

  8. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  9. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    YongliangZhang; ChenDong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses. Cellular & Molecular Immunology. 2005;2(1):20-27.

  10. Recent innovations in mRNA vaccines.

    Science.gov (United States)

    Ulmer, Jeffrey B; Geall, Andrew J

    2016-08-01

    Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have yet been licensed for human use. Recently, mRNA based vaccines have emerged as an alternative approach. They promise the flexibility of plasmid DNA vaccines, without the need for electroporation, but with enhanced immunogenicity and safety. In addition, they avoid the limitations of anti-vector immunity seen with viral vectors, and can be dosed repeatedly. This review highlights the key papers published over the past few years and summarizes prospects for the near future.

  11. Alternative polyadenylation of mRNA precursors

    Science.gov (United States)

    Tian, Bin; Manley, James L.

    2017-01-01

    Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

  12. Molecular characterization of adenosine 5'-monophosphate deaminase--the key enzyme responsible for the umami taste of nori (Porphyra yezoensis Ueda, Rhodophyta).

    Science.gov (United States)

    Minami, Seiko; Sato, Minoru; Shiraiwa, Yoshihiro; Iwamoto, Koji

    2011-12-01

    The enzyme adenosine 5'-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5'-monophosphate to inosine 5'-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.

  13. Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5'-monophosphate.

    Science.gov (United States)

    Shi, Fan; Gong, Shixing; Xu, Li; Zhu, Huanhuan; Sun, Zhenfan; Sun, Wei

    2013-12-01

    In this paper, a graphene (GR) ionic liquid (IL) 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite-modified carbon molecular wire electrode (CMWE) was fabricated by a drop-casting method and further applied to the sensitive electrochemical detection of adenosine-5'-monophosphate (AMP). CMWE was prepared with diphenylacetylene (DPA) as the modifier and the binder. The properties of modified electrode were examined by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical behaviors of AMP was carefully investigated with enhanced responses appeared, which was due to the presence of GR-IL composite on the electrode surface with excellent electrocatalytic ability. A well-defined oxidation peak of AMP appeared at 1.314 V and the electrochemical parameters were calculated by electrochemical methods. Under the selected conditions, the oxidation peak current of AMP was proportional to its concentration in the range from 0.01 μM to 80.0 μM with the detection limit as 3.42 nM (3σ) by differential pulse voltammetry. The proposed method exhibited good selectivity and was applied to the detection of vidarabine monophosphate injection samples with satisfactory results.

  14. Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases.

    Science.gov (United States)

    Linka, R M; Risse, S L; Bienemann, K; Werner, M; Linka, Y; Krux, F; Synaeve, C; Deenen, R; Ginzel, S; Dvorsky, R; Gombert, M; Halenius, A; Hartig, R; Helminen, M; Fischer, A; Stepensky, P; Vettenranta, K; Köhrer, K; Ahmadian, M R; Laws, H-J; Fleckenstein, B; Jumaa, H; Latour, S; Schraven, B; Borkhardt, A

    2012-05-01

    The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.

  15. Domain compatibility in Ire1 kinase is critical for the unfolded protein response.

    Science.gov (United States)

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J; Tirasophon, Witoon

    2010-07-16

    The unfolded protein response is a mechanism to cope with endoplasmic reticulum stress. In Saccharomyces cerevisiae, Ire1 senses the stress and mediates a signaling cascade to upregulate responsive genes through an unusual HAC1 mRNA splicing. The splicing requires interconnected activity (kinase and endoribonuclease (RNase)) of Ire1 to cleave HAC1 mRNA at the non-canonical splice sites before translation into Hac1 transcription factor. Analysis of the truncated kinase domain from Ire1 homologs revealed that this domain is highly conserved. Characterization by domain swapping indicated that a functional ATP/ADP binding domain is minimally required. However the overall domain compatibility is critical for eliciting its full RNase function.

  16. Proteolytic susceptibility of creatine kinase isozymes and arginine kinase.

    Science.gov (United States)

    Ercan, Altan; Grossman, Steven H

    2003-07-11

    The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.

  17. Identification of lung cancer oncogenes based on the mRNA expression and single nucleotide polymorphism profile data.

    Science.gov (United States)

    Wang, Y; Mei, Q; Ai, Y Q; Li, R Q; Chang, L; Li, Y F; Xia, Y X; Li, W H; Chen, Y

    2015-01-01

    This study aimed to identify the oncogenes associated with lung cancer based on the mRNA and single nucleotide polymorphism (SNP) profile data. The mRNA expression profile data of GSE43458 (80 cancer and 30 normal samples) and SNP profile data of GSE33355 (61 pairs of lung cancer samples and control samples) were downloaded from Gene Expression Omnibus database. Common genes between the mRNA profile and SNP profile were identified as the lung cancer oncogenes. Risk subpathways of the selected oncogenes with the SNP locus were analyzed using the iSubpathwayMiner package in R. Moreover, protein-protein interaction (PPI) network of the oncogenes was constructed using the HPRD database and then visualized using the Cytoscape. Totally, 3004 DEGs (1105 up-regulated and 1899 down-regulated) and 125 significant SNPs closely related to 174 genes in the lung cancer samples were identified. Also, 39 common genes, like PFKP (phosphofructokinase, platelet) and DGKH-rs11616202 (diacylglycerol kinase, eta) that enriched in sub-pathways such as galactose metabolism, fructose and mannose metabolism, and pentose phosphate pathway, were identified as the lung cancer oncogenes. Besides, PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1), RORA (RAR-related orphan receptor A), MAGI3 (membrane associated guanylate kinase, WW and PDZ domain containing 3), PTPRM (protein tyrosine phosphatase, receptor type, M), and BMP6 (bone morphogenetic protein 6) were the hub genes in PPI network. Our study suggested that PFKP and DGKH that enriched in galactose metabolism, fructose and mannose metabolism pathway, as well as PIK3R1, RORA, and MAGI3, may be the lung cancer oncogenes.

  18. THOC5, a member of the mRNA export complex: a novel link between mRNA export machinery and signal transduction pathways in cell proliferation and differentiation.

    Science.gov (United States)

    Tran, Doan D H; Koch, Alexandra; Tamura, Teruko

    2014-01-10

    Cell growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of growth factor/cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation have been mainly studied by focusing on the cis- and trans-elements in promoters however, the role of mRNA export machinery during differentiation has not been adequately examined. THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5) is a member of THO complex which is a subcomplex of the transcription/export complex (TREX). THOC5 is evolutionarily conserved in higher eukaryotes, however the exact roles of THOC5 in transcription and mRNA export are still unclear. In this review, we focus on recently uncovered aspects of the role of THOC5 in signal transduction induced by extracellular stimuli. THOC5 is phosphorylated by several protein kinases at multiple residues upon extracellular stimuli. These include stimulation with growth factors/cytokines/chemokines, or DNA damage reagents. Furthermore, THOC5 is a substrate for several oncogenic tyrosine kinases, suggesting that THOC5 may be involved in cancer development. Recent THOC5 knockout mouse data reveal that THOC5 is an essential element in the maintenance of stem cells and growth factor/cytokine-mediated differentiation/proliferation. Furthermore, depletion of THOC5 influences less than 1% of total mRNA export in the steady state, however it influences more than 90% of growth factor/cytokine induced genes. THOC5, thereby contributes to the 3' processing and/or export of immediate-early genes induced by extracellular stimuli. These studies bring new insight into the link between the mRNA export complex and immediate-early gene response. The data from these studies also suggest that THOC5 may be a useful tool for studying stem cell biology, for modifying the differentiation processes and for cancer therapy.

  19. Akt pathway activation and increased neuropeptide Y mRNA expression in the rat hippocampus: implications for seizure blockade.

    Science.gov (United States)

    Goto, Eduardo M; Silva, Marcelo de Paula; Perosa, Sandra R; Argañaraz, Gustavo A; Pesquero, João B; Cavalheiro, Esper A; Naffah-Mazzacoratti, Maria G; Teixeira, Vicente P C; Silva, José A

    2010-04-01

    The aim of this study was to analyze the expression of survival-related molecules such Akt and integrin-linked kinase (ILK) to evaluate Akt pathway activation in epileptogenesis process. Furthermore, was also investigated the mRNA expression of neuropeptide Y, a considered antiepileptic neuropeptide, in the pilocarpine-induced epilepsy. Male Wistar rats were submitted to the pilocarpine model of epilepsy. Hippocampi were removed 6h (acute phase), 12h (late acute), 5d (silent) and 60d (chronic) after status epilepticus (SE) onset, and from animals that received pilocarpine but did not develop SE (partial group). Hippocampi collected were used to specify mRNA expression using Real-Time PCR. Immunohistochemistry assay was employed to place ILK distribution in the hippocampus and Western blot technique was used to determine Akt activation level. A decrease in ILK mRNA content was found during acute (0.39+/-0.03) and chronic (0.48+/-0.06) periods when compared to control group (0.87+/-0.10). Protein levels of ILK were also diminished during both periods. Partial group showed increased ILK mRNA expression (0.80+/-0.06) when compared with animals in the acute stage. Silent group had ILK mRNA and immunoreactivity similar to control group. Western blot assay showed an augmentation in Akt activation in silent period (0.52+/-0.03) in comparison with control group (0.44+/-0.01). Neuropeptide Y mRNA expression increased in the partial group (1.67+/-0.22) and in the silent phase (1.45+/-0.29) when compared to control group (0.36+/-0.12). Results suggest that neuropeptide Y (as anticonvulsant) might act in protective mechanisms occurred during epileptic phenomena. Together with ILK expression and Akt activation, these molecules could be involved in hippocampal neuroprotection in epilepsy. Copyright 2009 Elsevier Ltd. All rights reserved.

  20. Citron kinase - renaissance of a neglected mitotic kinase.

    Science.gov (United States)

    D'Avino, Pier Paolo

    2017-05-15

    Cell division controls the faithful segregation of genomic and cytoplasmic materials between the two nascent daughter cells. Members of the Aurora, Polo and cyclin-dependent (Cdk) kinase families are known to regulate multiple events throughout cell division, whereas another kinase, citron kinase (CIT-K), for a long time has been considered to function solely during cytokinesis, the last phase of cell division. CIT-K was originally proposed to regulate the ingression of the cleavage furrow that forms at the equatorial cortex of the dividing cell after chromosome segregation. However, studies in the last decade have clarified that this kinase is, instead, required for the organization of the midbody in late cytokinesis, and also revealed novel functions of CIT-K earlier in mitosis and in DNA damage control. Moreover, CIT-K mutations have recently been linked to the development of human microcephaly, and CIT-K has been identified as a potential target in cancer therapy. In this Commentary, I describe and re-evaluate the functions and regulation of CIT-K during cell division and its involvement in human disease. Finally, I offer my perspectives on the open questions and future challenges that are necessary to address, in order to fully understand this important and yet unjustly neglected mitotic kinase. © 2017. Published by The Company of Biologists Ltd.

  1. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    of the membrane-bound 1ß1 Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase 1 subunit derepressed GCN4 mRNA translation up to 70-fold. GCN4 translation was very sensitive to the presence of heterologous protein......This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2 (eIF-2 ) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4......, as a density of 1 of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation...

  2. ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes.

    Science.gov (United States)

    Ellederová, Zdenka; Cais, Ondrej; Susor, Andrej; Uhlírová, Katka; Kovárová, Hana; Jelínková, Lucie; Tomek, Wolfgang; Kubelka, Michal

    2008-02-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.

  3. Suppression of prostaglandin E(2)-mediated c-fos mRNA induction by interleukin-4 in murine macrophages.

    Science.gov (United States)

    Zhuang, D; Kawajiri, H; Takahashi, Y; Yoshimoto, T

    2000-03-01

    When murine peritoneal macrophages were stimulated for 30 min with arachidonic acid, the growth-associated immediate early gene c-fos was induced in a concentration-dependent manner as assessed by Northern blot analysis. The arachidonic acid-induced c-fos mRNA expression was inhibited by a cyclooxygenase inhibitor, indomethacin, but not by a lipoxygenase inhibitor, nordihydroguaiaretic acid. Macrophages produced prostaglandin (PG) E(2) from arachidonic acid as determined by an enzyme immunoassay. Northern blot analysis revealed the expression of PGE receptor EP2 and EP4 subtypes, but not EP1 and EP3 in murine macrophages. PGE(2) brought about a marked elevation of cAMP, and c-fos mRNA expression was increased by PGE(2) and dibutyryl cAMP in these cells. These results suggest that arachidonic acid is transformed to PGE(2), which then binds to EP2 and EP4 receptors to increase intracellular cAMP and c-fos mRNA expression. Furthermore, the induction of c-fos by arachidonic acid, PGE(2), and cAMP was suppressed by pretreatment with interleukin (IL)-4. We also showed that the tyrosine phosphorylation of a Janus kinase, JAK3, is enhanced by IL-4 treatment, suggesting that the PGE(2)-mediated c-fos mRNA induction is inhibited by IL-4 through the tyrosine phosphorylation of JAK3.

  4. Integrated Analysis of DNA Methylation and mRNA Expression Profiles to Identify Key Genes in Severe Oligozoospermia

    Directory of Open Access Journals (Sweden)

    Zhiming Li

    2017-05-01

    Full Text Available Severe oligozoospermia (SO is a complex disorder, whose etiology is the combined effect of genetic factors and epigenetic conditions. In this study, we examined DNA methylation and mRNA expression status in a set of testicular tissues of SO patients (n = 3, and compared methylated data with those derived from obstructive azoospermia (OA patients (n = 3 with normal spermatogenesis phenotype. We identified 1,960 differentially methylated CpG sites showing significant alterations in SO vs. OA using the Illumina Infinium HumanMethylation450 bead array. By integrating above DNA methylation data and mRNA expression results, we totally identified 72 methylated CpG sites located in 65 genes with anti-correlation between DNA methylation and mRNA expression. Integrated pathways analysis indicates that these genes are involved in response to hormone stimulus, activation of protein kinase activity, and apoptotic process, among others. We also observed some genes with inversely correlated difference is novel in male infertility field, including PTPRN2, EPHX1, SERPINB9, SLIT3, etc. Our results lay a groundwork for further biological study of SO. Moreover, we generated a workflow for integrated analysis of DNA methylation and mRNA expression, which is expandable to other study types.

  5. A network of PUF proteins and Ras signaling promote mRNA repression and oogenesis in C. elegans.

    Science.gov (United States)

    Hubstenberger, Arnaud; Cameron, Cristiana; Shtofman, Rebecca; Gutman, Shiri; Evans, Thomas C

    2012-06-15

    Cell differentiation requires integration of gene expression controls with dynamic changes in cell morphology, function, and control. Post-transcriptional mRNA regulation and signaling systems are important to this process but their mechanisms and connections are unclear. During C. elegans oogenesis, we find that two groups of PUF RNA binding proteins (RNABPs), PUF-3/11 and PUF-5/6/7, control different specific aspects of oocyte formation. PUF-3/11 limits oocyte growth, while PUF-5/6/7 promotes oocyte organization and formation. These two PUF groups repress mRNA translation through overlapping but distinct sets of 3' untranslated regions (3'UTRs). Several PUF-dependent mRNAs encode other mRNA regulators suggesting both PUF groups control developmental patterning of mRNA regulation circuits. Furthermore, we find that the Ras-MapKinase/ERK pathway functions with PUF-5/6/7 to repress specific mRNAs and control oocyte organization and growth. These results suggest that diversification of PUF proteins and their integration with Ras-MAPK signaling modulates oocyte differentiation. Together with other studies, these findings suggest positive and negative interactions between the Ras-MAPK system and PUF RNA-binding proteins likely occur at multiple levels. Changes in these interactions over time can influence spatiotemporal patterning of tissue development.

  6. The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability.

    Science.gov (United States)

    Chiaberge, S; Cassarino, E; Mangiarotti, G

    1998-10-16

    AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

  7. Effects of arsenic poisoning on neuronal cell apoptosis and mRNA and protein expression of calpain 1,calpain 2,and cdk5/p25

    Institute of Scientific and Technical Information of China (English)

    李新

    2014-01-01

    Objective To study the effect of arsenic on neuronal cell apoptosis and the mRNA and protein expression of calpain 1,calpain 2,and cyclin-dependent kinases 5(cdk5)/p25 and to provide a scientific basis for the research on neurotoxic mechanism of arsenic trioxide(As2O3).Methods Primary cultured rat neurons were divided into untreated control group,dimethyl sulfoxide

  8. Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells.

    Science.gov (United States)

    Parameswaran, Narayanan; Hall, Carolyn S; Bock, Barbara C; Sparks, Harvey V; Gallo, Kathleen A; Spielman, William S

    2002-12-01

    Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [3H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [3H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.

  9. WNK kinases and essential hypertension.

    Science.gov (United States)

    Huang, Chou-Long; Kuo, Elizabeth; Toto, Robert D

    2008-03-01

    The present review summarizes recent literature and discusses the potential roles of WNKs in the pathogenesis of essential hypertension. WNKs (with-no-lysine [K]) are a recently discovered family of serine-threonine protein kinases with unusual protein kinase domains. The role of WNK kinases in the control of blood pressure was first revealed by the findings that mutations of two members, WNK1 and WNK4, cause Gordon's syndrome. Laboratory studies have revealed that WNK kinases play important roles in the regulation of sodium and potassium transport. Animal models have been created to unravel the pathophysiology of sodium transport disorders caused by mutations of the WNK4 gene. Potassium deficiency causes sodium retention and increases hypertension prevalence. The expression of WNK1 is upregulated by potassium deficiency, raising the possibility that WNK1 may contribute to salt-sensitive essential hypertension associated with potassium deficiency. Associations of polymorphisms of WNK genes with essential hypertension in the general population have been reported. Mutations of WNK1 and WNK4 cause hypertension at least partly by increasing renal sodium retention. The role of WNK kinases in salt-sensitive hypertension within general hypertension is suggested, but future work is required to firmly establish the connection.

  10. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    Energy Technology Data Exchange (ETDEWEB)

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  11. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknot...

  12. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol

    2015-07-01

    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  13. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Directory of Open Access Journals (Sweden)

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  14. Electronic band structure and Fermi surfaces of the quasi-two-dimensional monophosphate tungsten bronze, P4W12O44

    Science.gov (United States)

    Paul, S.; Ghosh, A.; Sato, T.; Sarma, D. D.; Takahashi, T.; Wang, E.; Greenblatt, M.; Raj, S.

    2014-02-01

    The electronic structure of quasi-two-dimensional monophosphate tungsten bronze, P4W12O44, has been investigated by high-resolution angle-resolved photoemission spectroscopy and density functional theoretical calculations. Experimental electron-like bands around \\Gamma point and Fermi surfaces have similar shapes as predicted by calculations. Fermi surface mapping at different temperatures shows a depletion of density of states at low temperature in certain flat portions of the Fermi surfaces. These flat portions of the Fermi surfaces satisfy the partial nesting condition with incommensurate nesting vectors q_1 and q_2 , which leads to the formation of charge density waves in this phosphate tungsten bronzes. The setting up of charge density wave in these bronzes can well explain the anomaly observed in its transport properties.

  15. Dielectric spectra broadening as a signature for dipole-matrix interaction. III. Water in adenosine monophosphate/adenosine-5'-triphosphate solutions.

    Science.gov (United States)

    Puzenko, Alexander; Levy, Evgeniya; Shendrik, Andrey; Talary, Mark S; Caduff, Andreas; Feldman, Yuri

    2012-11-21

    In this, the third part of our series on the dielectric spectrum symmetrical broadening of water, we consider the nucleotide aqueous solutions. Where in Parts I [E. Levy et al., J. Chem. Phys. 136, 114502 (2012)] and II [E. Levy et al., J. Chem. Phys. 136, 114503 (2012)], the dipole-dipole or ion-dipole interaction had a dominant feature, now the interplay between these two types of dipole-matrix interactions will be considered. We present the results of high frequency dielectric measurements of different concentrations of adenosine monophosphate/adenosine-5'-triphosphate aqueous solutions. We observed the Cole-Cole broadening of the main relaxation peak of the solvent in the solutions. Moreover, depending on the nucleotide concentration, we observed both types of dipole-matrix interaction. The 3D trajectory approach (described in detail in Part I) is applied in order to highlight the differences between the two types of interaction.

  16. Effect of dietary fluorine from Araxá rock phosphate on the hepatic production of cyclic-adenosine monophosphate in broilers

    Directory of Open Access Journals (Sweden)

    Rezende M.J.M.

    1999-01-01

    Full Text Available The cyclic adenosine 3?, 5?-monophosphate (cAMP production was evaluated in liver thin sections of broiler chicks fed on a experimental diet containing bicalcium phosphate or Araxá rock phosphate (ARP as source of P, with a high content of fluorine, at different ages: from the first to the 42nd and from the 21st to the 42nd day of age. The intake of the ARP formulated diet starting from birth elicited an increase of cAMP production in broiler liver. However, when this diet was offered after the 21st day of age, the hepatic cAMP production in broilers was not significantly (P>0.05 affected, suggesting that the effect of high fluorine present in Araxá rock phosphate, on hepatic cAMP of broiler chicks depends on the age in which the experimental diet is started.

  17. Evolution of coherent collective modes through consecutive charge-density-wave transitions in the (PO2)4(WO3)12 monophosphate tungsten bronze

    Science.gov (United States)

    Stojchevska, L.; Borovšak, M.; Foury-Leylekian, P.; Pouget, J.-P.; Mertelj, T.; Mihailovic, D.

    2017-07-01

    All-optical femtosecond relaxation dynamics in a single crystal of monophosphate tungsten bronze (PO2)4(WO3)2m with alternate stacking m =6 of WO3 layers was studied through the three consequent charge-density-wave (CDW) transitions. Several transient coherent collective modes associated with the different CDW transitions were observed and analyzed in the framework of the time-dependent Ginzburg-Landau theory. Remarkably, the interference of the modes leads to an apparent rectification effect in the transient reflectivity response. A saturation of the coherent-mode amplitudes with increasing pump fluence well below the CDWs destruction threshold fluence indicates a decoupling of the electronic and lattice parts of the order parameter on the femtosecond timescale.

  18. Behavior of the monophosphate tungsten bronzes (PO{sub 2}){sub 4}(WO{sub 3}){sub 2m} (m=4 and 6) in electrochemical lithium insertion

    Energy Technology Data Exchange (ETDEWEB)

    Longoria Rodriguez, F.E.; Martinez-de la Cruz, A.; Lopez Cuellar, E. [Division de Estudios de Posgrado, Facultad de Ingenieria Mecanica y Electrica, Universidad Autonoma de Nuevo Leon, Pedro de Alba s/n, Ciudad Universitaria, C.P. 66451, San Nicolas de los Garza, NL (Mexico)

    2006-10-06

    The electrochemical lithium insertion process has been studied in the family of monophosphate tungsten bronzes (PO{sub 2}){sub 4}(WO{sub 3}){sub 2m}, where m=4 and 6. Structural changes in the pristine oxides were followed as lithium insertion proceeded. Through potentiostatic intermittent technique, the different processes which take place in the cathode during the discharge of the cell were analysed. The nature of the bronzes Li{sub x}(PO{sub 2}){sub 4}(WO{sub 3}){sub 2m} formed was determined by in situ X-ray diffraction experiments. These results have allowed establishment of a correlation with the reversible/irreversible processes detected during the electrochemical lithium insertion. Measurements of resistivity showed that upon lithium insertion, the metallic pristine oxides become insulating. (author)

  19. Investigation on the occurrence and significance of cyclic adenosine 3':5'-monophosphate in phytoplankton and natural aquatic communities

    Energy Technology Data Exchange (ETDEWEB)

    Francko, D.A.

    1980-01-01

    This study demonstrates, on the basis of several analyanalytical criteria, that the production and extracellular release of cyclic adenosine 3':5'-monophosphate (cAMP) is widespread among phytoplankton species. The production and release of CAMP varied markedly among different species grown under similar environmental conditions, and intraspecifically during the life cycle of a given algal species. This investigation marks the first time cAMP has been investigated in natural aquatic systems. An examination of epilimnetic lakewater samples from Lawrence Lake, a hardwater oligotrophic lake, and Wintergreen Lake, a hardwater hypereutrophic lake, both in southwestern Michigan, demonstrated that cAMP existed in both particulate-associated and dissolved forms in these systems.

  20. Changes of learning, memory and levels of CaMKII, CaM mRNA, CREB mRNA in the hippocampus of chronic multiple-stressed rats

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background The effect of chronic stress on cognitive functions has been one of the hot topic in neuroscience. But there has been much controversy over its mechanism. Such single stressor applied in the past could not simulate complicated living circumstances that people confronted with. The aim of this study was to investigate the effects of chronic multiple-stress on learning and memory as well as on the levels of calcium/calmodulin-dependent protein kinase II (CaMKII), calmodulin (CaM) mRNA, and cAMP-response element binding protein (CREB) mRNA in the hippocampus of rats. Methods The rats were divided randomly into stressed and control groups. The stressed group was given chronic multiple-stress for 6 weeks to set up a chronic multiple-stressed model. The rats' performance of spatial learning and memory was tested using Morris Water Maze (MWM) and Y-maze. Meanwhile, the expressions of CaMKII, CaM mRNA and CREB mRNA of rats' hippocampus were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the width of synaptic cleft and the thickness of post-synaptic densities (PSD) were observed in the hippocampal CA3 region of rats by electron microscopy. Results After exposure to chronic multiple-stress for 6 weeks, the ability of learning and memory of the stressed group was higher than that of the control group (P<0.05, P<0.01). The width of synaptic cleft was smaller and the thickness of PSD was larger in the hippocampal CA3 region of the stressed group than in that of the control group (P<0.01). The CaMKII immunostaining of the stressed group was stronger than that of the control group in the stratum radiatum and oriens of the hippocampal CA1 and CA3, especially in the stratum oriens. Quantitative analysis indicated that the expression of CaMKII, CaM mRNA, and CREB mRNA in the hippocampus of the stressed group was higher than that of the control group (P<0.05, P<0.01). Conclusions The

  1. Probing dimensionality beyond the linear sequence of mRNA.

    Science.gov (United States)

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions.

  2. Determination of SGK1 mRNA in non-small cell lung cancer samples underlines high expression in squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    Abbruzzese Claudia

    2012-01-01

    Full Text Available Abstract Background Lung cancer represents the most frequent cause of death for cancer. In non-small cell lung cancer (NSCLC, which accounts for the vast majority of this disease, only early detection and treatment, when possible, may significantly affect patient's prognosis. An important role in NSCLC malignancy is attributed to the signal transduction pathways involving PI3Kinase, with consequent activation of the AKT family factors. The serum and glucocorticoid kinase (SGK factors, which share high structural and functional homologies with the AKT factors, are a family of ubiquitously expressed serine/threonine kinases under the control of cellular stress and hormones. SGK1 is the most represented SGK member. Methods By means of immunohistochemistry and quantitative real-time PCR, we determined SGK1 protein and mRNA expression in a cohort of 66 formalin-fixed, paraffin-embedded NSCLC surgical samples. All samples belonged to patients with a well-documented clinical history. Results mRNA expression was significantly higher in squamous cell carcinomas, and correlated with several clinical prognostic indicators, being elevated in high-grade tumors and in tumors with bigger size and worse clinical stage. No correlation was found between SGK1 protein expression and these clinical parameters. Conclusions This explorative analysis of SGK1 expression in NSCLC samples highlights the potential role of this factor in NSCLC patients' prognosis. Moreover, the higher expression in the squamous cell carcinoma subtype opens new therapeutic possibilities in this NSCLC subtype by designing specific kinase inhibitors.

  3. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    Science.gov (United States)

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  4. Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Snyder Jeanne M

    2002-10-01

    Full Text Available Abstract Background It has been proposed that high insulin levels may cause delayed lung development in the fetuses of diabetic mothers. A key event in lung development is the production of adequate amounts of pulmonary surfactant. Insulin inhibits the expression of surfactant protein A (SP-A, the major surfactant-associated protein, in lung epithelial cells. In the present study, we investigated the signal transduction pathways involved in insulin inhibition of SP-A gene expression. Methods H441 cells, a human lung adenocarcinoma cell line, or human fetal lung explants were incubated with or without insulin. Transcription run-on assays were used to determine SP-A gene transcription rates. Northern blot analysis was used to examine the effect of various signal transduction inhibitors on SP-A gene expression. Immunoblot analysis was used to evaluate the levels and phosphorylation states of signal transduction protein kinases. Results Insulin decreased SP-A gene transcription in human lung epithelial cells within 1 hour. Insulin did not affect p44/42 mitogen-activated protein kinase (MAPK phosphorylation and the insulin inhibition of SP-A mRNA levels was not affected by PD98059, an inhibitor of the p44/42 MAPK pathway. In contrast, insulin increased p70 S6 kinase Thr389 phosphorylation within 15 minutes. Wortmannin or LY294002, both inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase, or rapamycin, an inhibitor of the activation of p70 S6 kinase, a downstream effector in the PI 3-kinase pathway, abolished or attenuated the insulin-induced inhibition of SP-A mRNA levels. Conclusion Insulin inhibition of SP-A gene expression in lung epithelial cells probably occurs via the rapamycin-sensitive PI 3-kinase signaling pathway.

  5. Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals.

    Science.gov (United States)

    Gao, Xueliang; Wang, Haizhen; Yang, Jenny J; Chen, Jing; Jie, Jiang; Li, Liangwei; Zhang, Yinwei; Liu, Zhi-Ren

    2013-05-31

    Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

  6. Synergistic effect of vasoactive intestinal peptides on TNF-alpha-induced IL-6 synthesis in osteoblasts: amplification of p44/p42 MAP kinase activation.

    Science.gov (United States)

    Natsume, Hideo; Tokuda, Haruhiko; Mizutani, Jun; Adachi, Seiji; Matsushima-Nishiwaki, Rie; Minamitani, Chiho; Kato, Kenji; Kozawa, Osamu; Otsuka, Takanobu

    2010-05-01

    We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of vasoactive intestinal peptide (VIP) on TNF-alpha-induced IL-6 synthesis in these cells. VIP, which by itself slightly stimulated IL-6 synthesis, synergistically enhanced the TNF-alpha-induced IL-6 synthesis in MC3T3-E1 cells. The synergistic effect of VIP on the TNF-alpha-induced IL-6 synthesis was concentration-dependent in the range between 1 and 70 nM. We previously reported that VIP stimulated cAMP production in MC3T3-E1 cells. Forskolin, a direct activator of adenylyl cyclase, or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP), a plasma membrane-permeable cAMP analogue, markedly enhanced the TNF-alpha-induced IL-6 synthesis as well as VIP. VIP markedly up-regulated the TNF-alpha-induced p44/p42 MAP kinase phosphorylation. The Akt phosphorylation stimulated by TNF-alpha was only slightly affected by VIP. PD98059, a specific inhibitor of MEK1/2, significantly suppressed the enhancement of TNF-alpha-induced IL-6 synthesis by VIP. The synergistic effect of a combination of VIP and TNF-alpha on the phosphorylation of p44/p42 MAP kinase was diminished by H-89, an inhibitor of cAMP-dependent protein kinase. These results strongly suggest that VIP synergistically enhances TNF-alpha-stimulated IL-6 synthesis via up-regulating p44/p42 MAP kinase through the adenylyl cyclase-cAMP system in osteoblasts.

  7. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Muscle in Growing Pigs

    Directory of Open Access Journals (Sweden)

    X. M. Men

    2016-04-01

    Full Text Available The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA, to quantity myosin heavy-chains (MyHCs mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (% of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01, and MyHC I mRNA proportion was positively correlated with creatine kinase (CK, succinate dehydrogenase (SDH, malate dehydrogenase (MDH activities (p<0.05. These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics.

  8. Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

    Science.gov (United States)

    Autieri, M V; Fresa, K L; Coffman, F D; Katz, M E; Cohen, S

    1990-12-01

    We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.

  9. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine...

  10. Renal targeting of kinase inhibitors

    NARCIS (Netherlands)

    Dolman, M. E. M.; Fretz, M. M.; Segers, Gj. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J.

    2008-01-01

    Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tub

  11. Inhibitors of protein kinase C

    Institute of Scientific and Technical Information of China (English)

    LIU Shiying; JIANG Yuyang; CAO Jian; LIU Feng; MA Li; ZHAO Yufen

    2005-01-01

    Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups of protein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family. They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer, inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.

  12. Renal targeting of kinase inhibitors

    NARCIS (Netherlands)

    Dolman, M. E. M.; Fretz, M. M.; Segers, Gj. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J.

    2008-01-01

    Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within

  13. Deferasirox in pyruvate kinase deficiency

    OpenAIRE

    Deeren, Dries

    2008-01-01

    Deferasirox in pyruvate kinase deficiency phone: +32-51-237437 (Deeren, Dries) (Deeren, Dries) Department of Haematology, Heilig-Hartziekenhuis Roeselare-Menen vzw - Wilgenstraat 2 - B-8800 - Roeselare - BELGIUM (Deeren, Dries) BELGIUM Registration: 2008-09-10 Received: 2008-09-05 Accepted: 2008-09-10 ePublished: 2008-09-23

  14. Anticancer Alkaloid Lamellarins Inhibit Protein Kinases

    Directory of Open Access Journals (Sweden)

    Laurent Meijer

    2008-10-01

    Full Text Available Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dualspecificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.

  15. Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability

    Directory of Open Access Journals (Sweden)

    Gorospe Myriam

    2005-05-01

    Full Text Available Abstract Background Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. Results In order to investigate the relative contribution of gene transcription and changes in mRNA stability regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell and nuclear run-on (newly transcribed RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes. Conclusion We propose that regulation of mRNA stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

  16. Effects of DNA replication on mRNA noise.

    Science.gov (United States)

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories.

  17. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    OpenAIRE

    WANG Ling; Fisher, Laura A.; Wahl, James K.; Peng, Aimin

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that pla...

  18. Ligustrazine attenuates the platelet-derived growth factor-BB-induced proliferation and migration of vascular smooth muscle cells by interrupting extracellular signal-regulated kinase and P38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Yu, Lifei; Huang, Xiaojing; Huang, Kai; Gui, Chun; Huang, Qiaojuan; Wei, Bin

    2015-07-01

    The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) leads to intimal thickening of the aorta and is, therefore, important in the development of arteriosclerosis. As a result, the use of antiproliferative and antimigratory agents for VSMCs offers promise for the treatment of vascular disorders. Although several studies have demonstrated that ligustrazine may be used to treat heart and blood vessel diseases, the detailed mechanism underlying its actions remain to be elucidated. In the present study, the inhibitory effect of ligustrazine on platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation and migration, and the underlying mechanisms were investigated. The findings demonstrated that ligustrazine significantly inhibited PDGF-BB-stimulated VSMC proliferation. VSMCs dedifferentiated into a proliferative phenotype under PDGF-BB stimulation, which was effectively reversed by the administration of ligustrazine. In addition, ligustrazine also downregulated the production of nitric oxide and cyclic guanine monophosphate, induced by PDGF-BB. Additionally, ligustrazine significantly inhibited PDGF-BB-stimulated VSMC migration. Mechanistic investigation indicated that the upregulation of cell cycle-associated proteins and the activation of the extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (MAPK) signaling induced by PDGF-BB was suppressed by the administration of ligustrazine. In conclusion, the present study, demonstrated for the first time, to the best of our knowledge, that ligustrazine downregulated PDGF-BB-induced VSMC proliferation and migration partly, at least, through inhibiting the activation of the ERK and P38 MAPK signaling.

  19. AMP N1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

    Directory of Open Access Journals (Sweden)

    Noriko Hattori

    2010-01-01

    Full Text Available Earlier we identified adenosine monophosphate (AMP N1-oxide as a unique compound of royal jelly (RJ that induces neurite outgrowth (neuritegenesis from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK but also that of cAMP/calcium-response element-binding protein (CREB in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

  20. Thermodynamics of molecular recognition of mRNA 5' cap by yeast eukaryotic initiation factor 4E.

    Science.gov (United States)

    Kiraga-Motoszko, Katarzyna; Niedzwiecka, Anna; Modrak-Wojcik, Anna; Stepinski, Janusz; Darzynkiewicz, Edward; Stolarski, Ryszard

    2011-07-14

    Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpy–entropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5′-5′ triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution. © 2011 American Chemical Society

  1. Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor

    Science.gov (United States)

    Vijayaraj, Preethi; Kröger, Cornelia; Reuter, Ursula; Windoffer, Reinhard; Leube, Rudolf E.

    2009-01-01

    Keratin intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which affects cytoarchitecture, cell growth, survival, and organelle transport. However, owing to redundancy, the global function of keratins has not been defined in full. Using a targeted gene deletion strategy, we generated transgenic mice lacking the entire keratin multiprotein family. In this study, we report that without keratins, embryonic epithelia suffer no cytolysis and maintain apical polarity but display mislocalized desmosomes. All keratin-null embryos die from severe growth retardation at embryonic day 9.5. We find that GLUT1 and -3 are mislocalized from the apical plasma membrane in embryonic epithelia, which subsequently activates the energy sensor adenosine monophosphate kinase (AMPK). Analysis of the mammalian target of rapamycin (mTOR) pathway reveals that AMPK induction activates Raptor, repressing protein biosynthesis through mTORC1's downstream targets S6 kinase and 4E-binding protein 1. Our findings demonstrate a novel keratin function upstream of mTOR signaling via GLUT localization and have implications for pathomechanisms and therapy approaches for keratin disorders and the analysis of other gene families. PMID:19841136

  2. Protein kinase D1 deficiency promotes differentiation in epidermal keratinocytes

    Science.gov (United States)

    Choudhary, Vivek; Olala, Lawrence O.; Kaddour-Djebbar, Ismail; Helwa, Inas; Bollag, Wendy B.

    2014-01-01

    Background Protein kinase D (PKD or PKD1) is a serine/threonine protein kinase that has been shown to play a role in a variety of cellular processes; however, the function of PKD1 in the skin has not been fully investigated. The balance between proliferation and differentiation processes in the predominant cells of the epidermis, the keratinocytes, is essential for normal skin function. Objective To investigate the effect of PKD1 deficiency on proliferation and differentiation of epidermal keratinocytes. Methods We utilized a floxed PKD1 mouse model such that infecting epidermal keratinocytes derived from these mice with an adenovirus expressing Cre-recombinase allowed us to determine the effect of PKD1 gene loss in vitro. Proliferation and differentiation were monitored using qRT-PCR, Western blot, transglutaminase activity assays, [3H]thymidine incorporation into DNA and cell cycle analysis. Results A significant decrease in PKD1 mRNA and protein levels was achieved in adenoviral Cre-recombinase-infected cells. Deficiency of PKD1 resulted in significant increases in the mRNA and protein expression of various differentiation markers such as loricrin, involucrin, and keratin 10 either basally and/or upon stimulation of differentiation. PKD1-deficient keratinocytes also showed an increase in transglutaminase expression and activity, indicating an anti-differentiative role of PKD1. Furthermore, the PKD1-deficient keratinocytes exhibited decreased proliferation. However, PKD1 loss had no effect on stem cell marker expression. Conclusions Cre-recombinase-mediated knockdown represents an additional approach demonstrating that PKD1 is an anti-differentiative, pro-proliferative signal in mouse keratinocytes. PMID:25450094

  3. Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts

    NARCIS (Netherlands)

    Westra, J; Limburg, PC; de Boer, Peter; van Rijswijk, Martin

    2004-01-01

    Objective: To investigate the effect of the p38 mitogen activated protein kinase ( MAPK) inhibitor RWJ 67657 on inflammatory mediator production by rheumatoid synovial fibroblasts (RSF). Methods: RSF were pretreated with RWJ 67657 and stimulated with TNFalpha and/or IL-1beta. Protein levels and mRNA

  4. Lipid-soluble smoke particles upregulate vascular smooth muscle ETB receptors via activation of mitogen-activating protein kinases and NF-kappaB pathways

    DEFF Research Database (Denmark)

    Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei

    2008-01-01

    ), elevated levels of ET(B) receptor mRNA (quantitative real-time PCR), and protein expressions (immunohistochemistry and Western blotting). Intracellular signaling was studied with Western blotting and phosphoELISA; this revealed that DSP induced extracellular-regulated protein kinases 1 and 2 (ERK1/2), p38...

  5. Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

    OpenAIRE

    Fuller, Stephen J.; Osborne, Sally A.; Leonard, Sam J.; Hardyman, Michelle A.; Vaniotis, George; Allen, Bruce G.; Sugden, Peter H.; Clerk, Angela

    2015-01-01

    Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) d...

  6. Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

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    Christine S Hopp

    Full Text Available Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA, maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.

  7. Elucidating biological risk factors in suicide: role of protein kinase A.

    Science.gov (United States)

    Dwivedi, Yogesh; Pandey, Ghanshyam N

    2011-06-01

    Suicide is a major public health concern. Although there have been several studies of suicidal behavior that focused on the roles of psychosocial and sociocultural factors, these factors are of too little predictive value to be clinically useful. Therefore, research on the biological perspective of suicide has gained a stronghold and appears to provide a promising approach to identify biological risk factors associated with suicidal behavior. Recent studies demonstrate that an alteration in synaptic and structural plasticity is key to affective illnesses and suicide. Signal transduction molecules play an important role in such plastic events. Protein kinase A (PKA) is a crucial enzyme in the adenylyl cyclase signal transduction pathway and is involved in regulating gene transcription, cell survival, and plasticity. In this review, we critically and comprehensively discuss the role of PKA in suicidal behavior. Because stress is an important component of suicide, we also discuss whether stress affects PKA and how this may be associated with suicidal behavior. In addition, we also discuss the functional significance of the findings regarding PKA by describing the role of important PKA substrates (i.e., Rap1, cyclic adenosine monophosphate response element binding protein, and target gene brain-derived neurotrophic factor). These studies suggest the interesting possibility that PKA and related signaling molecules may serve as important neurobiological factors in suicide and may be relevant in target-specific therapeutic interventions for these disorders.

  8. mRNA trafficking and local translation: the Yin and Yang of regulating mRNA localization in neurons

    Institute of Scientific and Technical Information of China (English)

    John R. Sinnamon; Kevin Czaplinski

    2011-01-01

    Localized translation and the requisite trafficking of the mRNA template play significant roles in the nervous system including the establishment of dendrites and axons,axon path-finding,and synaptic plasticity.We provide a brief review on the regulation of localizing mRNA in mammalian neurons through critical posttranslational modifications of the factors involved.These examples highlight the relationship between mRNA trafficking and the translational regulation of trafficked mRNAs and provide insight into how extracellular signals target these events during signal transduction.

  9. Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP signaling to inhibit steroidogenesis in human granulosa cells

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    HoYuen Basil

    2009-10-01

    Full Text Available Abstract Background Obesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells. Methods The effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG cells was examined by Enzyme linked immunosorbent assay (ELISA. The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner. Results and Conclusion In the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059 and p38 (SB203580 inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone

  10. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  11. Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity

    Science.gov (United States)

    Miralem, Tihomir; Gibbs, Peter E. M.; Revert, Fernando; Saus, Juan; Maines, Mahin D.

    2010-01-01

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis. PMID:20177069

  12. Increased mRNA Levels of TCF7L2 and MYC of the Wnt Pathway in Tg-ArcSwe Mice and Alzheimer's Disease Brain

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    Elin S. Blom

    2011-01-01

    Full Text Available Several components in the Wnt pathway, including β-catenin and glycogen synthase kinase 3 beta, have been implied in AD pathogenesis. Here, mRNA brain levels from five-month-old tg-ArcSwe and nontransgenic mice were compared using Affymetrix microarray analysis. With surprisingly small overall changes, Wnt signaling was the most affected pathway with altered expression of nine genes in tg-ArcSwe mice. When analyzing mRNA levels of these genes in human brain, transcription factor 7-like 2 (TCF7L2 and v-myc myelocytomatosis viral oncogene homolog (MYC, were increased in Alzheimer's disease (AD (P<.05. Furthermore, no clear differences in TCF7L2 and MYC mRNA were found in brains with frontotemporal lobar degeneration, suggesting that altered regulation of these Wnt-related genes could be specific to AD. Finally, mRNA levels of three neurogenesis markers were analyzed. Increased mRNA levels of dihydropyrimidinase-like 3 were observed in AD brain, suggesting that altered Wnt pathway regulation may signify synaptic rearrangement or neurogenesis.

  13. Prognostic Impact of mRNA Expression Levels of HER1–4 (ERBB1–4 in Patients with Locally Advanced Rectal Cancer

    Directory of Open Access Journals (Sweden)

    Melanie Kripp

    2016-01-01

    Full Text Available Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC undergoing perioperative chemoradiotherapy (CRT. Members of the human epidermal growth factor receptor (HER family of receptor tyrosine kinases EGFR (HER1, ERBB1, HER2 (ERBB2, HER3 (ERBB3, and HER4 (ERBB4 are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC. Patients and Methods. ERBB1–4 mRNA expression and tumor proliferation using Ki-67 (MKI67 mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63 treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial. Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression of ERBB1 (EGFR, HER1 had significantly increased risk for recurrence and death. Patients with high mRNA expression of MKI67 had reduced risk for relapse. Conclusion. This analysis suggests a prognostic impact of both ERBB1 and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy.

  14. NS-187 (INNO-406, a Bcr-Abl/Lyn Dual Tyrosine Kinase Inhibitor

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    Tomoko Niwa

    2007-01-01

    Full Text Available Protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP to the hydroxyl groups of protein side chains, and they play critical roles in regulating cellular signal transduction and other biochemical processes. They are attractive targets for today’s drug discovery and development, and many pharmaceutical companies are intensively developing various kinds of protein kinase inhibitors. A good example is the recent success with the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (GleevecTM in the treatment of chronic myeloid leukemia. Though imatinib has dramatically improved the treatment of Bcr-Abl-positive chronic myeloid leukemia, resistance is often found in patients with advanced-stage disease. Several mechanisms have been proposed to explain this resistance, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA, increased drug efflux mediated by P-glycoprotein, and activation of the Src-family kinase (SFK Lyn. We set out to develop a novel drug whose affinity for Abl is higher than that of imatinib and whose specifi city in inhibiting Lyn is higher than that of SFK/Abl inhibitors such as dasatinib (SprycelTM or bosutinib (SKI-606. Our work has led to the development of NS-187 (INNO-406, a novel Abl/Lyn dual tyrosine kinase inhibitor with clinical prospects. To provide an overview of how a selective kinase inhibitor has been developed, this review presents chemical-modification studies carried out with the guidance of molecular modeling, the structural basis for the high potency and selectivity of NS-187 based on the X-ray structure of the NS-187/Abl complex, and the biological profi ling of NS-187, including site-directed mutagenesis experiments.

  15. HuD interacts with Bdnf mRNA and is essential for activity-induced BDNF synthesis in dendrites.

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    Filip Vanevski

    Full Text Available Highly specific activity-dependent neuronal responses are necessary for modulating synapses to facilitate learning and memory. We present evidence linking a number of important processes involved in regulating synaptic plasticity, suggesting a mechanistic pathway whereby activity-dependent signaling, likely through protein kinase C (PKC-mediated phosphorylation of HuD, can relieve basal repression of Bdnf mRNA translation in dendrites, allowing for increased TrkB signaling and synaptic remodeling. We demonstrate that the neuronal ELAV family of RNA binding proteins associates in vivo with several Bdnf mRNA isoforms present in the adult brain in an activity-dependent manner, and that one member, HuD, interacts directly with sequences in the long Bdnf 3' untranslated region (3'UTR and co-localizes with Bdnf mRNA in dendrites of hippocampal neurons. Activation of PKC leads to increased dendritic translation of mRNAs containing the long Bdnf 3'UTR, a process that is dependent on the presence of HuD and its phosphorylation at threonine residues 149 and/or 165. Thus, we found a direct effect of HuD on regulating translation of dendritic Bdnf mRNAs to mediate local and activity-dependent increases in dendritic BDNF synthesis.

  16. eIF2A mediates translation of hepatitis C viral mRNA under stress conditions.

    Science.gov (United States)

    Kim, Joon Hyun; Park, Sung Mi; Park, Ji Hoon; Keum, Sun Ju; Jang, Sung Key

    2011-05-10

    Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.

  17. Dihydrotestosterone regulating apolipoprotein M expression mediates via protein kinase C in HepG2 cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Ye

    2012-12-01

    Full Text Available Abstract Background Administration of androgens decreases plasma concentrations of high-density lipid cholesterol (HDL-C. However, the mechanisms by which androgens mediate lipid metabolism remain unknown. This present study used HepG2 cell cultures and ovariectomized C57BL/6 J mice to determine whether apolipoprotein M (ApoM, a constituent of HDL, was affected by dihydrotestosterone (DHT. Methods HepG2 cells were cultured in the presence of either DHT, agonist of protein kinase C (PKC, phorbol-12-myristate-13-acetate (PMA, blocker of androgen receptor flutamide together with different concentrations of DHT, or DHT together with staurosporine at different concentrations for 24 hrs. Ovariectomized C57BL/6 J mice were treated with DHT or vehicle for 7d or 14d and the levels of plasma ApoM and livers ApoM mRNA were measured. The mRNA levels of ApoM, ApoAI were determined by real-time RT-PCR. ApoM and ApoAI were determined by western blotting analysis. Results Addition of DHT to cell culture medium selectively down-regulated ApoM mRNA expression and ApoM secretion in a dose-dependent manner. At 10 nM DHT, the ApoM mRNA levels were about 20% lower than in untreated cells and about 40% lower at 1000 nM DHT than in the control cells. The secretion of ApoM into the medium was reduced to a similar extent. The inhibitory effect of DHT on ApoM secretion was not blocked by the classical androgen receptor blocker flutamide but by an antagonist of PKC, Staurosporine. Agonist of PKC, PMA, also reduced ApoM. At 0.5 μM PMA, the ApoM mRNA levels and the secretion of ApoM into the medium were about 30% lower than in the control cells. The mRNA expression levels and secretion of another HDL-associated apolipoprotein AI (ApoAI were not affected by DHT. The levels of plasma ApoM and liver ApoM mRNA of DHT-treated C57BL/6 J mice were lower than those of vehicle-treated mice. Conclusions DHT directly and selectively down-regulated the level of ApoM mRNA and the

  18. Endocytosis of Receptor Tyrosine Kinases

    Science.gov (United States)

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  19. Receptor tyrosine kinases in carcinogenesis.

    Science.gov (United States)

    Zhang, Xiao-Ying; Zhang, Pei-Ying

    2016-11-01

    Receptor tyrosine kinases (RTKs) are cell surface glycoproteins with enzymatic activity involved in the regulation of various important functions. In all-important physiological functions including differentiation, cell-cell interactions, survival, proliferation, metabolism, migration and signaling these receptors are the key players of regulation. Additionally, mutations of RTKs or their overexpression have been described in many human cancers and are being explored as a novel avenue for a new therapeutic approach. Some of the deregulated RTKs observed to be significantly affected in cancers included vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, RTK-like orphan receptor 1 (ROR1) and the platelet-derived growth factor receptor. These deregulated RTKs offer attractive possibilities for the new anticancer therapeutic approach involving specific targeting by monoclonal antibodies as well as kinase. The present review aimed to highlight recent perspectives of RTK ROR1 in cancer.

  20. Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization.

    Directory of Open Access Journals (Sweden)

    Joppe W R Hovius

    2008-02-01

    Full Text Available Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi-induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK-dependent decrease of IL-6 and TNF-alpha mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor-induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.

  1. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxybenzoic Acid

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    Kuan-Han Lee

    2014-01-01

    Full Text Available Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxybenzoic acid (TMPBA and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 µM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4',6-diamidino-2-phenylindole (DAPI nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP kinases, 5' adenosine monophosphate-activated protein kinase (AMPK, and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.

  2. The Arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes.

    Directory of Open Access Journals (Sweden)

    Stuart Meier

    Full Text Available BACKGROUND: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3',5'-cyclic monophosphate (cGMP, has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. PRINCIPAL FINDINGS: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10 as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10(431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. CONCLUSIONS: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP.

  3. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart

    2010-01-26

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  4. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  6. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  7. RIP Kinases Initiate Programmed Necrosis

    Institute of Scientific and Technical Information of China (English)

    Lorenzo Galluzzi; Oliver Kepp; Guido Kroemer

    2009-01-01

    Some lethal stimuli can induce either apoptosis or necrosis, depending on the cell type and/or experimental setting. Until recently,the molecular bases of this phenomenon were largely unknown. Now, two members of the receptor-interacting serine-threonine kinase (RIP) family, RIP1 and RIP3, have been demonstrated to control the switch between apoptotic and necrotic cell death.Some mechanistic details, however, remain controversial.

  8. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  9. Counteracting roles of AMP deaminase and AMP kinase in the development of fatty liver.

    Directory of Open Access Journals (Sweden)

    Miguel A Lanaspa

    Full Text Available Fatty liver (hepatic steatosis is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP. It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation -either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.

  10. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  11. Oxidized Form of Creatine Kinase

    Institute of Scientific and Technical Information of China (English)

    王希成; 王帆; 邹晓明; 周海梦

    1994-01-01

    The purified rabbit muscle creatine kinase (R-CK) was previously considered homogeneousand without disulfide bonds.By the method of NR/R two-dimensional diagonal SDS-PAGE,two forms of R-CK,designated respectively "oxidized form" of creatine kinase which contained intrachain disulfide bondsand "reduced form" of creatine kinase which did not have any —S—S— bridges,were for the first time sepa-rated.They were found to be the same in amino acid composition,in subunit molecular Weight and in isoelec-tric point,and were almost identical in enzyme activities.Thus it is hard to isolate one from the other bycommon biochemical methods.More extensive studies show that the oxidized form of CK also contains a pair of reactive thiol groupswhich are essential to the enzyme activity,and it has one intrachain disulfide bond per subunit.In the nativestate,this —S—S— bond cannot be reduced by DTT,but by treating the reduced form of CK with some ox-idants,these —S—S— bonds can be formed in vitro.Thus it is presumed that the disulfide bonds are cross-linked through the oxidization of two shallowly buried —SH groups.

  12. Characterization of a protein kinase gene responsive to auxin and gibberellin in cucumber hypocotyls.

    Science.gov (United States)

    Chono, M; Nemoto, K; Yamane, H; Yamaguchi, I; Murofushi, N

    1998-09-01

    By means of the PCR, cDNA clones encoding putative protein kinases have been obtained from cucumber hypocotyls. The abundance of the transcript of one of these genes, which was named CsPK3, increased on treatment with gibberellin (GA4) and/or auxin (IAA). We screened a cucumber cDNA library to clone CsPK3 cDNA. The cDNA clone (cCsPK3) encodes an open reading frame of 1,413 bp (471 amino acids), and its predicted amino acid sequence showed homology with those of serine/threonine protein kinases. Northern blot analysis indicated that IAA was more active than GA4 in increasing the level of CsPK3 mRNA in cucumber hypocotyls and that the increase in the level of CsPK3 mRNA on treatment with IAA was not inhibited by pretreatment with a protein synthesis inhibitor. The level of CsPK3 mRNA was high in hypocotyls of dark-grown cucumber seedlings and decreased to less than 50% of the original level within 15 min of the start of irradiation with white light.

  13. TRPV4 regulates insulin mRNA expression and INS-1E cell death via ERK1/2 and NO-dependent mechanisms.

    Science.gov (United States)

    Billert, M; Skrzypski, M; Sassek, M; Szczepankiewicz, D; Wojciechowicz, T; Mergler, S; Strowski, M Z; Nowak, K W

    2017-03-27

    TRPV4 is a Ca(2+)-permeable, nonselective cation channel. Recently, TRPV4 was implicated in controlling peripheral insulin sensitivity, insulin secretion and apoptosis of pancreatic beta cells. Here, we characterize the role and potential mechanisms of TRPV4 in regulating insulin mRNA expression and cell death in insulin producing INS-1E cells and rat pancreatic islets. TRPV4 protein production was downregulated by siRNA. Intracellular calcium level was measured using Fluo-3 AM. Gene expression was studied by real-time PCR. Phosphorylation of extracellular signal-regulated kinase (ERK1 and ERK2) was detected by Western blot. Nitric oxide (NO) production was assessed by chemiluminescent reaction. Reactive oxygen species (ROS) level was analysed using a fluorogenic dye (DCFDA). Cell death was evaluated by determination of cytoplasmic histone-associated DNA fragments. Downregulation of TRPV4 neither affected insulin mRNA expression nor INS-1E cell growth. By contrast, pharmacological TRPV4 activation by 100nmol/l GSK1016790A increased Ca(2+) levels in INS-1E cells and enhanced insulin mRNA expression after 1 and 3h, whereas a suppression of both was detected after 24h incubation. GSK1016790A increased ERK1/2 phosphorylation and NO production but not ROS production. Pharmacological blockade of ERK1/2 attenuated GSK1016790A-induced insulin mRNA expression. Inhibition of NO synthesis by l-NAME failed to affect insulin mRNA expression in GSK1016790A treated INS-1E cells. Furthermore, inhibition of NO production attenuated GSK1016790A-induced INS-1E cell death. In pancreatic islets, 100nmol/l GSK1016790A increased insulin mRNA levels after 3h without inducing cytotoxicity after 24h. In conclusion, TRPV4 differently regulates insulin mRNA expression in INS-1E cells via ERK1/2 and NO-dependent mechanisms.

  14. The effects of ropivacaine hydrochloride on the expression of CaMK II mRNA in the dorsal root ganglion neurons.

    Science.gov (United States)

    Wen, Xianjie; Lai, Xiaohong; Li, Xiaohong; Zhang, Tao; Liang, Hua

    2016-12-01

    In this study, we identified the subtype of Calcium/calmodulin-dependent protein kinase II (CaMK II) mRNA in dorsal root ganglion neurons and observed the effects of ropivacaine hydrochloride in different concentration and different exposure time on the mRNA expression. Dorsal root ganglion neurons were isolated from the SD rats and cultured in vitro. The mRNA of the CaMK II subtype in dorsal root ganglion neurons were detected by real-time PCR. As well as, the dorsal root ganglion neurons were treated with ropivacaine hydrochloride in different concentration (1mM,2mM, 3mM and 4mM) for the same exposure time of 4h, or different exposure time (0h,2h,3h,4h and 6h) at the same concentration(3mM). The changes of the mRNA expression of the CaMK II subtype were observed with real-time PCR. All subtype mRNA of the CaMK II, CaMK IIα, CaMK IIβ, CaMK II δ, CaMK IIγ, can be detected in dorsal root ganglion neurons. With the increased of the concentration and exposure time of the ropivacaine hydrochloride, all the subtype mRNA expression increased. Ropivacaine hydrochloride up-regulate the CaMK IIβ, CaMK IIδ, CaMK IIg mRNA expression with the concentration and exposure time increasing. The nerve blocking or the neurotoxicity of the ropivacaine hydrochloride maybe involved with CaMK II. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  16. Sphingosine kinase 2-deficiency mediated changes in spinal pain processing

    Directory of Open Access Journals (Sweden)

    Jastrow eCanlas

    2015-08-01

    Full Text Available Chronic pain is one of the most burdensome health issues facing the planet (as costly as diabetes and cancer combined, and in desperate need for new diagnostic targets leading to better therapies. The bioactive lipid sphingosine 1-phosphate (S1P and its receptors have recently been shown to modulate nociceptive signalling at the level of peripheral nociceptors and central neurons. However, the exact role of S1P generating enzymes, in particular sphingosine kinase 2 (Sphk2, in nociception remains unknown. We found that both sphingosine kinases, Sphk1 and Sphk2, were expressed in spinal cord with higher levels of Sphk2 mRNA compared to Sphk1. All three Sphk2 mRNA-isoforms were present with the Sphk2.1 mRNA showing the highest relative expression. Mice deficient in Sphk2 (Sphk2-/- showed in contrast to mice deficient in Sphk1 (Sphk1-/- substantially lower spinal S1P levels compared to wild-type C57BL/6 mice. In the formalin model of acute peripheral inflammatory pain, Sphk2-/- mice showed facilitation of nociceptive transmission during the late response, whereas responses to early acute pain, and the number of c-Fos immunoreactive dorsal horn neurons were not different between Sphk2-/- and wild-type mice. Chronic peripheral inflammation (CPI caused a bilateral increase in mechanical sensitivity in Sphk2-/- mice. Additionally, CPI increased the relative mRNA expression of P2X4 receptor, brain-derived neurotrophic factor and inducible nitric oxide synthase in the ipsilateral spinal cord of wild-type but not Sphk2-/- mice. Similarly, Sphk2-/- mice showed in contrast to wild-type no CPI-dependent increase in areas of the dorsal horn immunoreactive for the microglia marker Iba-1 and the astrocyte marker GFAP. Our results suggest that the tightly regulated cell signalling enzyme Sphk2 may be a key component for facilitation of nociceptive circuits in the CNS leading to central sensitization and pain memory formation.

  17. Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie

    2014-05-01

    The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

  18. Proinflammatory cytokines, IL-1β and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts.

    Science.gov (United States)

    Eda, Hiroyuki; Shimada, Hideaki; Beidler, David R; Monahan, Joseph B

    2011-11-01

    The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1β (17-fold) and TNF-α (74-fold). IL-1β and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1β- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1β- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1β and TNF-α by up to 7- and 11-fold, respectively. IL-1β- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1β and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.

  19. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    Science.gov (United States)

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

  20. Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes

    DEFF Research Database (Denmark)

    Ye, Q; Wei, Y; Fischer, R

    1997-01-01

    kinase C using Northern blot analysis with three CaM gene specific cDNA probes. Five species of CaM mRNA were detected in all these cells. Surprisingly many of the investigated cell lines exhibited a decreased content of all CaM mRNAs as compared to control cells with CaMI and CaMII transcripts showing...

  1. Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner.

    Science.gov (United States)

    Moran, Stewart T; Haider, Khaleda; Ow, Yongkai; Milton, Peter; Chen, Luojing; Pillai, Shiv

    2003-06-13

    Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.

  2. The use of online heart-cutting high-performance liquid chromatography coupled with linear ion trap mass spectrometry in the identification of impurities in vidarabine monophosphate.

    Science.gov (United States)

    Wang, Hang; Xu, Tongzhou; Yuan, Jiaojian

    2017-02-17

    It is difficult to identify unknown impurities in nucleotide analogues by mass spectrometry because mass-spectrometry-incompatible mobile phases need to be used to separate the major ingredient from impurities. In this study, vidarabine monophosphate was selected, and unknown impurities were identified by online heart-cutting two-dimensional high-performance liquid chromatography and linear ion trap mass spectrometry. The one-dimensional reversed-phase column was filled with a mobile phase containing nonvolatile salt. In two-dimensional high-performance liquid chromatography, we used an Acclaim Q1 column with volatile salt, and the detection wavelength was 260 nm. The mass spectrum was scanned in positive- and negative-ion mode. The online heart-cutting and online demineralization technique ensured that the mobile phase was compatible with mass spectrometry; seven impurities were identified by MS(2) and MS(3) fragments. The mass fragmentation patterns of these impurities were investigated. The two isomers were semiprepared and complemented by nuclear magnetic resonance. The results were further compared with those of normal-phase high-performance liquid chromatography with mass spectrometry. The online heart-cutting two-dimensional high-performance liquid chromatography with mass spectrometry was superior in identifying more impurities. The method solves the problem of incompatibility between the mobile phase and mass spectrometry, so it is suitable for identifying unknown impurities. This method may also be used for investigating impurities in other nucleotide analogues.

  3. Symmetry and twins in the monophosphate tungsten bronze series (P02)4(W03)2m (2 < or = m < or = 14)

    Science.gov (United States)

    Roussel; Labbe; Groult

    2000-06-01

    Monophosphate tungsten bronze with pentagonal tunnels (PO2)4(WO3)2m are low-dimensional materials with charge density wave (CDW)-type electron instabilities. Two forms of the structure can thus be expected for all the members of the series: a low-temperature form (LT) corresponding to the CDW state and a high-temperature form (HT) corresponding to a normal metallic state. The HT form is described here for m = 9 and compared with that of the m = 5 and m = 7 counterparts. It is shown that a systematic twin phenomenon must be taken into account for HT members because of two possible configurations of the tilting mode of WO6 octahedra, The structure is also compared with that of m = 10, which exhibits the modulated CDW-LT form at room temperature. Owing to two possible polarization directions of the segments built of m WO6 octahedra, a twin phenomenon is also encountered in the LT forms. A review of all the structures known at present (m = 2, 4, 5, 6, 7, 8, 9, 10, 12) leads us to propose a structural law based on the building mode of W06 octahedra in W03-type slabs to explain the symmetry changes observed between even and odd members of the series.

  4. Measuring the dynamics of cyclic adenosine monophosphate level in living cells induced by low-level laser irradiation using bioluminescence resonance energy transfer

    Science.gov (United States)

    Huang, Yimei; Zheng, Liqin; Yang, Hongqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen; Zeng, Haishan

    2015-05-01

    Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors' expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI.

  5. Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning

    Science.gov (United States)

    GAO, Hanchao; ZHAO, Chengjiang; XIANG, Xi; LI, Yong; ZHAO, Yanli; LI, Zesong; PAN, Dengke; DAI, Yifan; HARA, Hidetaka; COOPER, David K.C.; CAI, Zhiming; MOU, Lisha

    2016-01-01

    Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications. PMID:27725344

  6. Modification of Tau by 8-Nitroguanosine 3',5'-Cyclic Monophosphate (8-Nitro-cGMP): EFFECTS OF NITRIC OXIDE-LINKED CHEMICAL MODIFICATION ON TAU AGGREGATION.

    Science.gov (United States)

    Yoshitake, Jun; Soeda, Yoshiyuki; Ida, Tomoaki; Sumioka, Akio; Yoshikawa, Misato; Matsushita, Kenji; Akaike, Takaaki; Takashima, Akihiko

    2016-10-21

    Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid β peptide deposition are hallmarks of Alzheimer's disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimer's disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Association study of three single-nucleotide polymorphisms in the cyclic adenosine monophosphate response element binding 1 gene and major depressive disorder.

    Science.gov (United States)

    Wei, Yange; Bu, Shufang; Liu, Xican; Li, Hengfen

    2015-06-01

    Major depressive disorder is a common chronic emotional disorder, and cyclic adenosine monophosphate response element binding protein 1 (CREB1) is hypothesized to play a role in its pathogenesis. The aim of the present study was to investigate the associations between major depressive disorder and relevant single nucleotide polymorphisms (SNPs) in the CREB1 gene. A total of 1,038 subjects of Han Chinese descent were recruited, including 456 patients with major depressive disorder (case group) and 582 healthy volunteers (control group). The frequency distributions of the genotypes and alleles were estimated in the case and control groups, and analyzed for any correlation with major depressive disorder. Three relevant SNP sites in CREB1 were analyzed using quantitative polymerase chain reaction, and statistical analyses were performed to estimate their use as risk factors for major depressive disorder. The analyses revealed that rs2254137 and rs16839883 in CREB1 showed polymorphisms in the sample population, and the genotype and allele frequencies of rs16839883 differed significantly when comparing the patients and healthy controls (P0.05). Furthermore, no statistically significant differences were detected in rs2254137 genotype and allele distribution when comparing the male and female patients with their corresponding control groups (P>0.05). However, statistically significant differences were observed in the genotype and allele frequencies of rs16839883 when the male and female patients were compared with their respective controls (Pmajor depressive disorder, which suggests that this SNP site should be further studied as a potential biomarker for major depressive disorder.

  8. Cyclic 3',5'-adenosine monophosphate (cAMP) signaling in the anterior pituitary gland in health and disease.

    Science.gov (United States)

    Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Stratakis, Constantine A

    2017-08-16

    The cyclic 3',5'-adenosine monophosphate (cAMP) was the first among the so-called "second messengers" to be described. It is conserved in most organisms and functions as a signal transducer by mediating the intracellular effects of multiple hormones and neurotransmitters. In this review, we first delineate how different members of the cAMP pathway ensure its correct compartmentalization and activity, mediate the terminal intracellular effects, and allow the crosstalk with other signaling pathways. We then focus on the pituitary gland, where cAMP exerts a crucial function by controlling the responsiveness of the cells to hypothalamic hormones, neurotransmitters and peripheral factors. We discuss the most relevant physiological functions mediated by cAMP in the different pituitary cell types, and summarize the defects affecting this pathway that have been reported in the literature. We finally discuss how a deregulated cAMP pathway is involved in the pathogenesis of pituitary disorders and how it affects the response to therapy. Copyright © 2017. Published by Elsevier B.V.

  9. 从RNA酶解液中分离5'-尿苷酸%Separation of Uridine 5'-Monophosphate from the Enzymatic Degradation Solution of RNA

    Institute of Scientific and Technical Information of China (English)

    李德莹; 丁庆豹

    2011-01-01

    采用2根强酸性阳离子交换柱、1根弱碱性阴离子交换柱和1根强碱性阴离子交换柱进行4柱串联,可以从RNA酶解液中分离得到5'-尿苷酸,而不混有其它核苷酸,并对离子交换树脂种类、树脂量、洗脱剂等作了进一步研究.结果表明,采用4柱串联分离5'-尿苷酸,其总收率达到92.1%、结晶纯度达到86%以上.%Uridine 5'-monophosphate(UMP) could be separated from the enzymatic degradation solution of RNA with two strong acid cation exchange columns, one weak base anionic exchange column and one strong alkali anionic exchange column. UMP Obtained was not contaminated with other three ribonucleotides. The total yield of UMP achieved 92. 1% and the purity of UMP crystal reached above 86%. The type of ion exchange resin, resin amount and eluent were further studied.

  10. Investigation on the occurrence and significance of cyclic adenosine 3':5'-monophosphate in phytoplankton and natural aquatic communities

    Energy Technology Data Exchange (ETDEWEB)

    Francko, D.A.

    1980-01-01

    This study is an investigation into the occurrence and potential functions of cyclic adenosine 3':5'-monophosphate (cAMP), a potent and ubiquitous metabolic regulatory molecule in heterotrophic organisms, in phytoplankton and in natural aquatic communities. Laboratory-cultured phytoplankton were grown under both optimal and suboptimal nutrient regimes under constant temperature and illumination regimes. Cellular and extracellular cAMP production, characterized by a number of biochemical techniques, was correlated with growth rate dynamics, chlorophyll a synthesis, /sup 14/C-bicarbonate uptake, alkaline phosphatase activity, and heterocyst formation. The blue-green alga Anabaena flos-aquae was used as a model system in the examination of these metabolic variables. Additionally, this alga was used to test the effects of perturbation of cAMP levels on the aforementioned metabolic variables. Investigations on the occurrence and seasonal dynamics of cAMP in aquatic systems were conducted on Lawrence Lake, a hardwater oligotrophic lake, and on Wintergreen Lake, a hardwater hypereutrophic lake, both in southwestern Michigan. Putative cAMP from both systems was characterized by several biochemical techniques. Weekly sampling of particulate and dissolved cAMP in the epilimnia of both lakes was correlated with data on the rates of primary productivity, alkaline phosphatase activity, chlorophyll a synthesis and changes in phytoplankton community structure.

  11. Structural Basis of Differential Ligand Recognition by Two Classes of bis-(3-5)-cyclic Dimeric Guanosine Monophosphate-binding Riboswitches

    Energy Technology Data Exchange (ETDEWEB)

    K Smith; C Shanahan; E Moore; A Simon; S Strobel

    2011-12-31

    The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbone is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.

  12. Solubility of disodium cytidine 5′-monophosphate in different binary mixtures from 288.15 K to 313.15 K

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jin [College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing (China); National Engineering Technique Research Center for Biotechnology, Nanjing (China); Ma, Tianle; Li, An [National Engineering Technique Research Center for Biotechnology, Nanjing (China); State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing (China); Chen, Xiaochun; Chen, Yong; Xie, Jingjing [College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing (China); National Engineering Technique Research Center for Biotechnology, Nanjing (China); Wu, Jinglan, E-mail: yinghanjie@njut.edu.cn [College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing (China); National Engineering Technique Research Center for Biotechnology, Nanjing (China); Ying, Hanjie [College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing (China); National Engineering Technique Research Center for Biotechnology, Nanjing (China); State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing (China)

    2013-08-10

    Highlights: • Solubility of 5′-CMPNa{sub 2} in different systems was measured the first time. • Experimental data were correlated by CNIBS/Redlich–Kister model and Apelblat model. • Good agreement has been observed between the calculated and the experimental data. • Enthalpy and entropy were calculated by the van’t Hoff equation and Gibbs equation. - Abstract: The solubility of disodium cytidine 5′-monophosphate (5′-CMPNa{sub 2}) in methanol + water and ethanol + water binary mixtures was measured experimentally at the temperatures ranging from 288.15 to 313.15 K. The results showed that the solubility of 5′-CMPNa{sub 2} increased with the increasing of temperature and the mole fraction of water in different binary mixtures. The (CNIBS)/Redlich–Kister model and the semi-empirical Apelblat model were applied for the prediction of the experimental data. Both models could give satisfactory simulation results. In addition, the thermodynamic properties of the dissolution process such as Gibbs energy, enthalpy, and entropy were calculated using the van’t Hoff equation and the Gibbs equation. The results indicated that the dissolution process was endothermic.

  13. Application and optimization of the tenderization of pig Longissimus dorsi muscle by adenosine 5'-monophosphate (AMP) using the response surface methodology.

    Science.gov (United States)

    Deng, Shaoying; Wang, Daoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong

    2016-03-01

    Based on single factor experiments, NaCl concentration, adenosine 5'-monophosphate (AMP) concentration and temperature were selected as independent variables for a three-level Box-Behnken experimental design, and the shear force and cooking loss were response values for regression analysis. According to the statistical models, it showed that all independent variables had significant effects on shear force and cooking loss, and optimal values were at the NaCl concentration of 4.15%, AMP concentration of 22.27 mmol/L and temperature of 16.70°C, which was determined with three-dimensional response surface diagrams and contour plots. Under this condition, the observed shear force and cooking loss were 0.625 kg and 8.07%, respectively, exhibiting a good agreement with their predicted values, showing the good applicability and feasibility of response surface methodology (RSM) for improving pork tenderness. Compared with control pig muscles, AMP combined with NaCl treatment demonstrated significant effects on improvement of meat tenderness and reduction of cooking loss. Therefore, AMP could be regarded as an effective tenderization agent for pork.

  14. 5′-Adenosine Monophosphate-Induced Hypothermia Attenuates Brain Ischemia/Reperfusion Injury in a Rat Model by Inhibiting the Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Yi-Feng Miao

    2015-01-01

    Full Text Available Hypothermia treatment is a promising therapeutic strategy for brain injury. We previously demonstrated that 5′-adenosine monophosphate (5′-AMP, a ribonucleic acid nucleotide, produces reversible deep hypothermia in rats when the ambient temperature is appropriately controlled. Thus, we hypothesized that 5′-AMP-induced hypothermia (AIH may attenuate brain ischemia/reperfusion injury. Transient cerebral ischemia was induced by using the middle cerebral artery occlusion (MCAO model in rats. Rats that underwent AIH treatment exhibited a significant reduction in neutrophil elastase infiltration into neuronal cells and matrix metalloproteinase 9 (MMP-9, interleukin-1 receptor (IL-1R, tumor necrosis factor receptor (TNFR, and Toll-like receptor (TLR protein expression in the infarcted area compared to euthermic controls. AIH treatment also decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL- positive neuronal cells. The overall infarct volume was significantly smaller in AIH-treated rats, and neurological function was improved. By contrast, rats with ischemic brain injury that were administered 5′-AMP without inducing hypothermia had ischemia/reperfusion injuries similar to those in euthermic controls. Thus, the neuroprotective effects of AIH were primarily related to hypothermia.

  15. Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

    Directory of Open Access Journals (Sweden)

    Kenji Ezoe

    Full Text Available Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK or 3-isobutyl-1-methylxanthine (IBMX to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

  16. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

    Science.gov (United States)

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  17. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results.

  18. Optimal Down Regulation of mRNA Translation

    Science.gov (United States)

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2017-01-01

    Down regulation of mRNA translation is an important problem in various bio-medical domains ranging from developing effective medicines for tumors and for viral diseases to developing attenuated virus strains that can be used for vaccination. Here, we study the problem of down regulation of mRNA translation using a mathematical model called the ribosome flow model (RFM). In the RFM, the mRNA molecule is modeled as a chain of n sites. The flow of ribosomes between consecutive sites is regulated by n + 1 transition rates. Given a set of feasible transition rates, that models the outcome of all possible mutations, we consider the problem of maximally down regulating protein production by altering the rates within this set of feasible rates. Under certain conditions on the feasible set, we show that an optimal solution can be determined efficiently. We also rigorously analyze two special cases of the down regulation optimization problem. Our results suggest that one must focus on the position along the mRNA molecule where the transition rate has the strongest effect on the protein production rate. However, this rate is not necessarily the slowest transition rate along the mRNA molecule. We discuss some of the biological implications of these results. PMID:28120903

  19. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    Science.gov (United States)

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  20. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    Science.gov (United States)

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  1. Significance and Expression of Serum and Glucocorticoid-inducible Kinase in Kidney of Mice with Diabetic Nephropathy

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression and the role of three isoforms of Serum and Glucocorticoidinducible Kinase (SGK) in experimental diabetic nephropathy (DN), 12 male C57BL/6 mice of 8-weeks-old were divided into two groups. Streptozotocin (STZ)-induced diabetic nephropathy and normal controls were analyzed at the end of the 4th week after the induction of diabetes. Renal hemodynamics and histological studies were performed. The expression of SGK1 mRNA, SGK2 mRNA and SGK3 mRNA of kidney cortex were measured by RT-PCR, and the cortical SGK1 protein was detected with Western blotting. Our results showed that the blood glucose, blood HbA1c, 24-h urinary protein, creatinine clearance and the renal index were all increased in DN group. More extracellular matrix (ECM) accumulation was observed. The level of cortical SGK1 mRNA and protein were up-regulated in DN group in comparison with control group. SGK2 and SGK3 mRNA were elevated in DN mice. In DN, mRNA level of three SGK isoforms and SGK1 protein were increased significantly. It is concluded that SGKs may contribute to the early renal injury of DN.

  2. LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

    Directory of Open Access Journals (Sweden)

    Yujie Zhang

    2016-03-01

    Full Text Available Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days. However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6, is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP, 25 kD FK506 binding protein (FKBP25 and RNA helicase A (RHA, contribute to this process.

  3. Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets

    Directory of Open Access Journals (Sweden)

    Jung-Hae Shin

    2015-10-01

    Conclusion: These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits [Ca2+]i mobilization in thrombin–platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

  4. Identification of tumor metastasis-related gene TMSG-1 by mRNA differential display

    Institute of Scientific and Technical Information of China (English)

    马春树; 刘宇欣; 郑杰; 方伟岗; 由江峰; 王洁良; 崔湘琳; 吴秉铨

    2002-01-01

    To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylation sites, 2 casein kinase II phosphorylation sites and 1 N-myristoylation site. The pattern of TMSG-1 expression in 6 types of human tumor tissues indicated levels of transcripts were the highest in prostate carcinoma. TMSG-1 had lower expression in metastases of lung carcinoma compared to primary lung carcinoma. Similarly the expression levels were higher in well-differentiated colon carcinoma than that in poorly differentiated colon carcinoma. TMSG-1 could also be detected in breast, ovarian, and pancreatic carcinoma. In 9 samples of primary gastric carcinoma tissues, RT-PCR and densitometric analysis demonstrated TMSG-1 expression levels in samples with lymph node metastases had a decreased tendency, compared to those without lymph node metastases. The difference was significant by student's t test (p<0.05). These results indicated TMSG-1 expression levels were inversely correlated with tumor metastatic potential.

  5. Protein Kinase D family kinases: roads start to segregate.

    Science.gov (United States)

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue.

  6. Elongation factor-2 phosphorylation in dendrites and the regulation of dendritic mRNA translation in neurons

    Directory of Open Access Journals (Sweden)

    Christopher eHeise

    2014-02-01

    Full Text Available Neuronal activity results in long lasting changes in synaptic structure and function by regulating mRNA translation in dendrites. These activity dependent events yield the synthesis of proteins known to be important for synaptic modifications and diverse forms of synaptic plasticity. Worthy of note, there is accumulating evidence that the eukaryotic Elongation Factor 2 Kinase (eEF2K/eukaryotic Elongation Factor 2 (eEF2 pathway may be strongly involved in this process. Upon activation, eEF2K phosphorylates and thereby inhibits eEF2, resulting in a dramatic reduction of mRNA translation. eEF2K is activated by elevated levels of calcium and binding of Calmodulin (CaM, hence its alternative name calcium/CaM-dependent protein kinase III (CaMKIII. In dendrites, this process depends on glutamate signaling and N-methyl-D-aspartate receptor (NMDAR activation. Interestingly, it has been shown that eEF2K can be activated in dendrites by the metabotropic glutamate receptor (mGluR 1/5 signaling, as well. Therefore, neuronal activity can induce local proteomic changes at the postsynapse by altering eEF2K activity. Well-established targets of eEF2K in dendrites include Brain-derived neurotrophic factor (BDNF, activity-regulated cytoskeletal-associated protein (Arc, the alpha subunit of calcium/CaM-dependent protein kinase II (αCaMKII, and Microtubule-associated protein 1B (MAP1B, all of which have well-known functions in different forms of synaptic plasticity.In this review we will give an overview of the involvement of the eEF2K/eEF2 pathway at dendrites in regulating the translation of dendritic mRNA in the context of altered NMDAR- and neuronal activity, and diverse forms of synaptic plasticity, such as metabotropic glutamate receptor-dependent-long-term depression (mGluR-LTD. For this, we draw on studies carried out both in vitro and in vivo.

  7. MAP kinases and histone modification

    Institute of Scientific and Technical Information of China (English)

    Tamaki Suganuma; Jerry L. Workman

    2012-01-01

    Signal transduction pathways alter the gene expression program in response to extracellular or intracellular cues.Mitogen-activated protein kinases (MAPKs) govern numerous cellular processes including cell growth,stress response,apoptosis,and differentiation.In the past decade,MAPKs have been shown to regulate the transcription machinery and associate with chromatin-modifying complexes.Moreover,recent studies demonstrate that several MAPKs bind directly to chromatin at target genes.This review highlights the recent discoveries of MAPK signaling in regard to histone modifications and chromatin regulation.Evidence suggesting that further unknown mechanisms integrate signal transduction with chromatin biology is discussed.

  8. Assessing protein kinase target similarity

    DEFF Research Database (Denmark)

    Gani, Osman A; Thakkar, Balmukund; Narayanan, Dilip

    2015-01-01

    : focussed chemical libraries, drug repurposing, polypharmacological design, to name a few. Protein kinase target similarity is easily quantified by sequence, and its relevance to ligand design includes broad classification by key binding sites, evaluation of resistance mutations, and the use of surrogate......" of sequence and crystal structure information, with statistical methods able to identify key correlates to activity but also here, "the devil is in the details." Examples from specific repurposing and polypharmacology applications illustrate these points. This article is part of a Special Issue entitled...

  9. MST kinases in development and disease

    OpenAIRE

    Thompson, Barry J.; Sahai, Erik

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cance...

  10. MST kinases in development and disease.

    Science.gov (United States)

    Thompson, Barry J; Sahai, Erik

    2015-09-14

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cancer, endothelial malformations, and autoimmune disease.

  11. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  12. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... properties are found in cancer cells. Investigation of these factors offers possibilities to understand the molecular background for TKl expression including to clarify general regulation patterns. It also gives valuable information for constructing new nucleoside analogs for the therapy of cancer and virus...... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  13. Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Nathanael S. (Berkeley, CA), Schultz, Peter (Oakland, CA), Wodicka, Lisa (Santa Clara, CA), Meijer, Laurent (Roscoff, FR), Lockhart, David J. (Mountain View, CA)

    2003-06-03

    The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

  14. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    Science.gov (United States)

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-05-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity.

  15. MAP kinase pathways and calcitonin influence CD44 alternate isoform expression in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Yang Kui

    2008-09-01

    Full Text Available Abstract Background Dysregulated expression and splicing of cell adhesion marker CD44 is found in many types of cancer. In prostate cancer (PC specifically, the standard isoform (CD44s has been found to be downregulated compared with benign tissue whereas predominant variant isoform CD44v7-10 is upregulated. Mitogen-activated protein kinase pathways and paracrine calcitonin are two common factors linked to dysregulated expression and splicing of CD44 in cancer. Calcitonin has been found to increase proliferation and invasion in PC acting through the protein kinase A pathway. Methods In androgen-independent PC with known high CD44v7-10 expression, CD44 total and CD44v7-10 RNA or protein were assessed in response to exogenous and endogenous calcitonin and to inhibitors of protein kinase A, MEK, JNK, or p38 kinase. Benign cells and calcitonin receptor-negative PC cells were also tested. Results MEK or p38 but not JNK reduced CD44 total RNA by 40%–65% in cancer and benign cells. Inhibition of protein kinase A reduced CD44 total and v7-10 protein expression. In calcitonin receptor-positive cells only, calcitonin increased CD44 variant RNA and protein by 3 h and persisting to 48 h, apparently dependent on an uninhibited p38 pathway. Cells with constitutive CT expression showed an increase in CD44v7-10 mRNA but a decrease in CD44 total RNA. Conclusion The MEK pathway increases CD44 RNA, while calcitonin, acting through the protein kinase A and p38 pathway, facilitates variant splicing. These findings could be used in the formulation of therapeutic methods for PC targeting CD44 alternate splicing.

  16. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

    Science.gov (United States)

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J; Andersson, Leif C; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-10-20

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy.

  17. The effects of pilates exercise on lipid metabolism and inflammatory cytokines mRNA expression in female undergraduates.

    Science.gov (United States)

    Kim, Hyo-Jin; Kim, Jiyeon; Kim, Chang-Sun

    2014-09-01

    The purpose of the study was to verify the effects of Pilates exercise by observing the impact of 8 weeks of Pilates exercise on lipid metabolism and inflammatory cytokine mRNA expression in female undergraduates in their 20s who had no prior experience in Pilates exercise and had not exercised in the previous 6 months. There were 18 subjects with no prior experience in Pilates exercise. The subjects were separated into the Pilates exercise group (n = 9) and the non-exercise control group (n = 9). The former performed Pilates exercise for 60-70 minutes over 8 weeks with a gradual strength increase of 9-16 in the Rating of Perceived Exercise (RPE). The body composition, creatine kinase in the bloodstream and lipid metabolism (TC, LDL-C, HDL-C, TG) were measured before and after the experiment and Real-Time PCR was used to investigate the mRNA expression of the inflammatory cytokines IL-6 and TNF-⍺. The creatine kinase (CK) in the blood had significant differences between the groups. The test group showed significant increase compared to the control group after 8 weeks of Pilates exercise (p = 0.007). Lipid analysis showed that the level of high-density lipoprotein cholesterol (HDL-C) was significantly different in the two groups (p = 0.049), with the Pilates exercise group exhibiting significantly higher levels compared to the control group. No significant differences were observed in the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG). IL-6 mRNA expression did not show significant differences between the groups either. Timing and TNF-α mRNA expression showed significant effect in both the exercise and the control groups (p = 0.013) but no correlation. It was found from the study that Pilates exercise for 8 weeks affected CK expression (the muscle damage marker) and induced positive changes in the levels of high-density lipoprotein.

  18. Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development.

    Science.gov (United States)

    Siemer, Corinna; Smiljakovic, Tatjana; Bhojwani, Monika; Leiding, Claus; Kanitz, Wilhelm; Kubelka, Michal; Tomek, Wolfgang

    2009-12-01

    Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed-loop model of translational initiation, we have analyzed components of the 5 -mRNA cap-binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E-BP1, and 3 -mRNA poly-(A) tail-associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16-cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA-binding factors occur at the transition from M II stage oocytes to 2-cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5 -end and 3 -end mRNA associated factors, mainly the poly-(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin-like protein with distinct eIF4E-binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16-cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by (7)m-GTP-Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development.

  19. Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos.

    Science.gov (United States)

    Saito, R; Fujita, N; Nagata, S

    2001-06-01

    The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.

  20. Focal Adhesion Kinase Regulates Expression of Thioredoxin-interacting Protein (TXNIP) in Cancer Cells

    OpenAIRE

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter ac...

  1. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    DEFF Research Database (Denmark)

    Brandauer, Josef; Vienberg, Sara Gry; Andersen, Marianne Agerholm

    2013-01-01

    for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependant. One-legged knee-extensor exercise training in humans increased Nampt protein......-activated protein kinase (AMPK) increases sirtuin activity by elevating NAD levels. As NAM directly inhibits sirtuins, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary...

  2. The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1

    NARCIS (Netherlands)

    Shah, K.; Russinova, E.; Gadella, T.W.J.; Willemse, J.; Vries, de S.C.

    2002-01-01

    The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does

  3. Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses.

    Science.gov (United States)

    Barth, Kenneth; Genco, Caroline Attardo

    2016-10-04

    The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses.

  4. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    Science.gov (United States)

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  5. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Jie Hong

    Full Text Available Mechanisms of the progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

  6. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

    Science.gov (United States)

    Hong, Jie; Li, Dan; Cao, Weibiao

    2016-01-01

    Mechanisms of the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

  7. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    NARCIS (Netherlands)

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  8. Deregulated Expression of Aurora Kinases Is Not a Prognostic Biomarker in Papillary Thyroid Cancer Patients

    Science.gov (United States)

    Prinzi, Natalie; Sorrenti, Salvatore; Falvo, Laura; De Vito, Corrado; Catania, Antonio; Tartaglia, Francesco; Mocini, Renzo; Coccaro, Carmela; Alessandrini, Stefania; Barollo, Susi; Mian, Caterina; Antonelli, Alessandro; De Antoni, Enrico; D’Armiento, Massimino; Ulisse, Salvatore

    2015-01-01

    A number of reports indicated that Aurora-A or Aurora-B overexpression represented a negative prognostic factor in several human malignancies. In thyroid cancer tissues a deregulated expression of Aurora kinases has been also demonstrated, butno information regarding its possible prognostic role in differentiated thyroid cancer is available. Here, weevaluated Aurora-A and Aurora-B mRNA expression and its prognostic relevance in a series of 87 papillary thyroid cancers (PTC), with a median follow-up of 63 months. The analysis of Aurora-A and Aurora-B mRNA levels in PTC tissues, compared to normal matched tissues, revealed that their expression was either up- or down-regulatedin the majority of cancer tissues. In particular, Aurora-A and Aurora-B mRNA levels were altered, respectively, in 55 (63.2%) and 79 (90.8%) out of the 87 PTC analyzed.A significant positive correlation between Aurora-A and Aurora-B mRNAswas observed (p=0.001). The expression of both Aurora genes was not affected by the BRAFV600E mutation. Univariate, multivariate and Kaplan-Mayer analyses documented the lack of association between Aurora-A or Aurora-B expression and clinicopathological parameterssuch as gender, age, tumor size, histology, TNM stage, lymph node metastasis and BRAF status as well asdisease recurrences or disease-free interval. Only Aurora-B mRNA was significantly higher in T(3-4) tissues, with respect to T(1-2) PTC tissues. The data reported here demonstrate that the expression of Aurora kinases is deregulated in the majority of PTC tissues, likely contributing to PTC progression. However, differently from other human solid cancers, detection of Aurora-A or Aurora-B mRNAs is not a prognostic biomarker inPTC patients. PMID:25807528

  9. Expression of deoxynucleoside kinases and 5'-nucleotidases in mouse tissues: implications for mitochondrial toxicity.

    Science.gov (United States)

    Rylova, Svetlana N; Mirzaee, Saeedeh; Albertioni, Freidoun; Eriksson, Staffan

    2007-06-30

    Anti-HIV nucleoside therapy can result in mitochondrial toxicity affecting muscles, peripheral nerves, pancreas and adipose tissue. The cytosolic deoxycytidine kinase (dCK; EC 2.7.1.74) and thymidine kinase (TK1; EC 2.7.1.21), the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK; EC 2.7.1.113) as well as 5'-deoxynucleotidases (5'-dNT; EC 3.1.3.5) are enzymes that control rate-limiting steps in formation of intracellular and intra-mitochondrial nucleotides. The mRNA levels and activities of these enzymes were determined in mouse tissues, using real-time PCR and selective enzyme assays. The expression of mRNA for all these enzymes and the mitochondrial deoxynucleotide carrier was detected in all tissues with a 5-10-fold variation. TK1 activities were only clearly detected in spleen and testis, while TK2, dGK and dCK activities were found in all tissues. dGK activities were higher than any other dNK in all tissues, except spleen and testis. In skeletal muscle dGK activity was 5-fold lower, TK2 and dCK levels were 10-fold lower as compared with other tissues. The variation in 5'-dNT activities was about eight-fold with the highest levels in brain and lowest in brown fat. Thus, the salvage of deoxynucleosides in muscles is 5-10-fold lower as compared to other non-proliferating tissues and 100-fold lower compared to spleen. These results may help to explain tissue specific toxicity observed with nucleoside analogs used in HIV treatment as well as symptoms in inherited mitochondrial TK2 deficiencies.

  10. Novel Library of Selenocompounds as Kinase Modulators

    Directory of Open Access Journals (Sweden)

    Carmen Sanmartín

    2011-07-01

    Full Text Available Although the causes of cancer lie in mutations or epigenic changes at the genetic level, their molecular manifestation is the dysfunction of biochemical pathways at the protein level. The 518 protein kinases encoded by the human genome play a central role in various diseases, a fact that has encouraged extensive investigations on their biological function and three dimensional structures. Selenium (Se is an important nutritional trace element involved in different physiological functions with antioxidative, antitumoral and chemopreventive properties. The mechanisms of action for selenocompounds as anticancer agents are not fully understood, but kinase modulation seems to be a possible pathway. Various organosulfur compounds have shown antitumoral and kinase inhibition effects but, in many cases, the replacement of sulfur by selenium improves the antitumoral effect of compounds. Although Se atom possesses a larger atomic volume and nucleophilic character than sulfur, Se can also formed interactions with aminoacids of the catalytic centers of proteins. So, we propose a novel chemical library that includes organoselenium compounds as kinase modulators. In this study thirteen selenocompounds have been evaluated at a concentration of 3 or 10 µM in a 24 kinase panel using a Caliper LabChip 3000 Drug Discover Platform. Several receptor (EGFR, IGFR1, FGFR1… and non-receptor (Abl kinases have been selected, as well as serine/threonine/lipid kinases (AurA, Akt, CDKs, MAPKs… implicated in main cancer pathways: cell cycle regulation, signal transduction, angiogenesis regulation among them. The obtained results showed that two compounds presented inhibition values higher than 50% in at least four kinases and seven derivatives selectively inhibited one or two kinases. Furthermore, three compounds selectively activated IGF-1R kinase with values ranging from −98% to −211%. In conclusion, we propose that the replacement of sulfur by selenium seems to be

  11. Transcriptional regulation of inhibin beta B messenger ribonucleic acid levels in TM.4 or primary rat Sertoli cells by 8-bromo-cyclic adenosine monophosphate.

    Science.gov (United States)

    Najmabadi, H; Rosenberg, L A; Yuan, Q X; Reyaz, G; Bhasin, S

    1993-04-01

    FSH, a major regulator of inhibin production in the testis, is believed to exert its effects via cAMP second messenger system. Inhibin alpha-subunit gene appears to be regulated by cAMP and has a palindromic cAMP response element sequence TGACGTCA. However, the regulation of the inhibin beta B-subunit gene by cAMP has been less clear. It has been assumed that beta B may not be regulated by cAMP, based mainly on observations that FSH stimulates only alpha, not beta B, mRNA levels, and that the 5'-up-stream regulatory region of the beta B gene does not contain the classical cAMP response element. However, we have observed that 8-bromo-cAMP stimulates beta B mRNA levels in both primary Sertoli (approximately 2-fold) and TM.4 cells (approximately 5-fold). We examined whether this cAMP-induced increase in beta B mRNA levels is the result of increased transcription or altered mRNA stability. Data from nuclear run-on assays demonstrate about a 2-fold increase in relative mRNA synthesis rates in primary Sertoli-cells and about a 4- to 5-fold increase in TM.4 cells. Transfection studies in TM.4 and JEG.3 cell lines with beta B:luciferase chimeric reporter gene constructs containing 1.5 kilobases of the beta B 5'-up-stream regulatory region revealed marked cAMP induction of reporter gene activity in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  13. Influenza virus mRNA trafficking through host nuclear speckles.

    Science.gov (United States)

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W; García-Sastre, Adolfo; Fontoura, Beatriz M A

    2016-05-27

    Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

  14. Growth hormone release from chicken anterior pituitary cells in primary culture: TRH and hpGRF synergy, protein synthesis, and cyclic adenosine 3'5'-monophosphate.

    Science.gov (United States)

    Perez, F M; Malamed, S; Scanes, C G

    1989-01-01

    Our earlier work showed that the effects of thyrotropin-releasing hormone (TRH) and human pancreatic growth hormone-releasing factor (hpGRF) on growth hormone (GH) release are synergistic (greater than additive) in a primary culture of chicken adenohypophyseal cells. The purpose of the present studies was to investigate the possible participation of protein synthesis and cyclic adenosine 3'5'-monophosphate (cAMP) in GH release. Following culture (48 hr), cells were incubated for 2 hr with test agents. Cycloheximide (an inhibitor of protein synthesis) had no effect on basal (absence of test agent) GH release or hpGRF-induced GH release. However, cycloheximide abolished the synergy between TRH and hpGRF. Although neither TRH nor hpGRF alone stimulated GH production (intracellular GH plus GH release) during a 2-hr incubation period, in combination these secretagogues increased total GH. These findings suggest that GH release from the chicken somatotroph under conditions of TRH and hpGRF synergy requires protein synthesis. In other studies, cells were exposed to agents inducing the formation of cAMP and either TRH or hpGRF. 8 Br-cAMP (10(-3) M), forskolin (10(-6) M), or isobutylmethylxanthine (IBMX; 10(-3) M) alone stimulated GH release to values between 30 and 50% over the basal value. The combined effects of each of these agents and TRH on GH release were synergistic. Similarly, IBMX and hpGRF exerted synergistic effects on GH release. In contrast, no synergy was shown between hpGRF and either 8 Br-cAMP or forskolin; their combined actions were less than additive.

  15. Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations.

    Science.gov (United States)

    Ueda, Atsushi; Wu, Chun-Fang

    2012-03-01

    Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss

  16. Conformational Changes in Orotidine 5’-Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5’-Phosphate Group Activates the Enzyme†

    Science.gov (United States)

    Desai, Bijoy J.; Wood, McKay; Fedorov, Alexander A.; Fedorov, Elena V.; Goryanova, Bogdana; Amyes, Tina L.; Richard, John P.; Almo, Steven C.; Gerlt, John A.

    2012-01-01

    The binding of a ligand to orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions are enforced that destabilize the carboxylate group of the substrate as well as stabilize the intermediate (in the Ec•S‡ complex). Focusing on the OMPDC from Methanothermobacter thermautotrophicus, the “remote” 5’-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that 1) directly contact the 5’-phosphate group; 2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from solvent; and 3) form hydrogen-bonding interactions across the interface between the “mobile” and “fixed” half-barrel domains of the (β/α8-barrel structure. Our data support a model in which the IBE provided by the 5’-phosphate group is used to enable interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec•S and Ec•S‡ complexes relative to the Eo•S complex. The conclusion that the IBE of the 5’-phosphate group provides stabilization of both the Ec•S and Ec•S‡ complexes, not just the Ec•S‡ complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions. PMID:23030629

  17. Cytidine-5'-monophosphate-N-acetylneuraminic acid. Asialoglycoprotein sialic acid transferase activity in liver and serum of patients with juvenile hepatic cirrhosis and alpha-1-antitrypsin deficiency.

    Science.gov (United States)

    Kuhlenschmidt, M S; Peters, S P; Pinkard, O D; Glew, R H; Sharp, H

    1976-04-08

    The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of

  18. Probing the Interaction between a DNA Nucleotide (Adenosine-5'-Monophosphate Disodium) and Surface Active Ionic Liquids by Rotational Relaxation Measurement and Fluorescence Correlation Spectroscopy.

    Science.gov (United States)

    Roy, Arpita; Banerjee, Pavel; Dutta, Rupam; Kundu, Sangita; Sarkar, Nilmoni

    2016-10-02

    This article demonstrates the interaction of a deoxyribonucleic acid (DNA) nucleotide, adenosine-5'-monophosphate disodium (AMP) with a cationic surface active ionic liquid (SAIL) 1-dodecyl-3-methylimidazoium chloride (C12mimCl) and an anionic SAIL, 1-butyl-3-methylimidazolium n-octylsulfate ([C4mim][C8SO4]). Dynamic light scattering (DLS) measurements and 1H NMR (nuclear magnetic resonance) studies indicate that substantial interaction is taking place among the DNA nucleotide, AMP and the SAILs. Moreover, cryogenic transmission electron microscopy (cryo-TEM) suggests that SAILs containing micellar assemblies are transformed into larger micellar assemblies in presence of DNA nucleotide. Additionally, the rotational motion of two oppositely charged molecules, Rhodamine 6G perchlorate (R6G) and Fluorescein sodium salt (Fl-Na) have been monitored in these aggregates. The rotational motion of R6G and Fl-Na differs significantly between SAILs micelles, and SAILs-AMP containing larger micellar aggregates. The effect of negatively charged DNA nucleotide (AMP) addition into the cationic and anionic SAILs is more prominent for the cationic charged molecule R6G than that of anionic probe Fl-Na due to the favourable electrostatic interaction between the AMP and cationic R6G. Moreover, the influence of the anionic DNA nucleotide on the cationic and anionic SAIL micelles is monitored through the variation of the lateral diffusion motion of oppositely charged probe molecules (R6G and Fl-Na) inside these aggregates. This variation in diffusion coefficient values also suggests that interaction pattern of these oppositely charged probes are different within the SAILs-nucleotide containing aggregates. Therefore, both rotational and translational diffusion measurements confirm that the DNA nucleotide (AMP) renders more rigid microenvironment within the micellar solution of SAILs.

  19. Mode-specific vibrational relaxation of photoexcited guanosine 5'-monophosphate and its acid form: a femtosecond broadband mid-IR transient absorption and theoretical study.

    Science.gov (United States)

    Zhang, Yuyuan; Improta, Roberto; Kohler, Bern

    2014-01-28

    UV-pump/broadband-mid-IR-probe transient absorption (TA) experiments and ab initio quantum mechanical (QM) calculations were used to investigate the photophysics in heavy water of the neutral and acid forms of guanosine 5'-monophosphate (GMP and GMPD(+), respectively). Excited GMP undergoes ultrafast internal conversion (IC) and returns to the electronic ground state in less than one picosecond with a large amount of excess vibrational energy. The spectroscopic signals are dominated by vibrational cooling - a process in which the solute dissipates vibrational energy to the solvent. For neutral GMP, cooling proceeds with a time constant of 3 ps. Following IC, at least some medium-frequency modes such as the carbonyl stretch and an in-plane ring vibration are excited, suggesting that the vibrational energy distribution is non-statistical. This is consistent with predicted structural changes upon passage through the S1/S0 conical intersection. GMPD(+) differs from GMP by a single deuteron at the N7 position, but has a dramatically longer lifetime of 200 ps. Vibrational cooling of the S1 state of GMPD(+) was monitored via several medium-frequency modes that were assigned using QM calculations. These medium-frequency modes are also vibrationally excited in a non-statistical fashion. Excitation of these modes is in line with the change in geometry at the S1 minimum of GMPD(+) predicted by QM calculations. Furthermore, these modes relax at different rates, fully consistent with QM calculations, which predict that excited vibrational states of the carbonyl stretch couple strongly to the D2O solvent and thus deactivate via intermolecular energy transfer (IET). In contrast, the ring stretch couples strongly to other ring modes of the guanine chromophore and appears to decay via intramolecular vibrational energy redistribution (IVR).

  20. Imipramine induces brain-derived neurotrophic factor mRNA expression in cultured astrocytes.

    Science.gov (United States)

    Takano, Katsura; Yamasaki, Hiroshi; Kawabe, Kenji; Moriyama, Mitsuaki; Nakamura, Yoichi

    2012-01-01

    Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.