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Sample records for oxide-cyclic guanosine monophosphate

  1. Involvement of nitric oxide-cyclic guanosine monophosphate pathway in the antidepressant-like effect of tropisetron and ondansetron in mice forced swimming test and tail suspension test.

    Science.gov (United States)

    Haj-Mirzaian, Arya; Kordjazy, Nastaran; Amiri, Shayan; Haj-Mirzaian, Arvin; Amini-Khoei, Hossien; Ostadhadi, Sattar; Dehpour, AhmadReza

    2016-06-05

    Antidepressant-like effects of 5-hydroxytryptamine subtype 3 (5-HT3) antagonists including tropisetron and ondansetron have been previously demonstrated in the literature. It was reported that stimulation of 5-HT3 receptors activate the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway, which is involved in regulation of behavioral and emotional functions. In our study, treating animals with tropisetron (5, 10, and 30mg/kg) and ondansetron (0.01 and 0.1µg/kg) significantly decreased the immobility time in forced swimming test (FST) and tail-suspension test (TST). Co-administration of subeffective doses of tropisetron (1mg/kg) and ondansetron (0.001µg/kg) with subeffective dose of l-NAME (10mg/kg, nonselective NO synthase (NOS) inhibitor) and 7-nitroindazole (25mg/kg, neural NOS inhibitor) exerted antidepressant-like effect in FST and TST, while aminoguanidine (50mg/kg, inducible NOS inhibitor) did not enhance the antidepressant-like effect of 5-HT3 antagonists. Besides, l-arginine (750mg/kg, NO precursor) and sildenafil (5mg/kg, phosphodiesterase inhibitor) suppressed the anti-immobility effect of 5-HT3 antagonists. None of the treatments altered the locomotor behavior of mice in open-field test. Also, hippocampal (but not cortical) nitrite level was significantly lower in tropisetron and ondansetron-treated mice compared with saline-injected mice. Also, co-administration of 7-nitroindazole with tropisetron or ondansetron caused a significant decrease in hippocampal nitrite levels. In conclusion, we suggest that antidepressant-like effect of tropisetron and ondansetron are partially mediated by modulation of NO-cGMP pathway. Copyright © 2016. Published by Elsevier B.V.

  2. The role of NMDA receptor and nitric oxide/cyclic guanosine monophosphate pathway in the antidepressant-like effect of dextromethorphan in mice forced swimming test and tail suspension test.

    Science.gov (United States)

    Sakhaee, Ehsan; Ostadhadi, Sattar; Khan, Muhammad Imran; Yousefi, Farbod; Norouzi-Javidan, Abbas; Akbarian, Reyhaneh; Chamanara, Mohsen; Zolfaghari, Samira; Dehpour, Ahmad-Reza

    2017-01-01

    Depression is a devastating disorder which has a high impact on the wellbeing of overall society. As such, need for innovative therapeutic agents are always there. Most of the researchers focused on N-methyl-d-aspartate receptor to explore the antidepressant like activity of new therapeutic agents. Dextromethorphan is a cough suppressant agent with potential antidepressant activity reported in mouse force swimming test. Considering N-methyl-d-aspartate as a forefront in exploring antidepressant agents, here we focused to unpin the antidepressant mechanism of dextromethorphan targeting N-methyl-d-aspartate receptor induced nitric oxide-cyclic guanosine monophosphate signaling. Dextromethorphan administered at a dose of 10 and 30mg/kg i.p significantly reduced the immobility time. Interestingly, this effect of drug (30mg/kg) was inhibited when the animals were pretreated either with N-methyl-d-aspartate (75mg/kg), or l-arginine (750mg/kg) as a nitric oxide precursor and/or sildenafil (5mg/kg) as a phosphodiesterase 5 inhibitor. However, the antidepressant effect of Dextromethorphan subeffective dose (3mg/kg) was augmented when the animals were administered with either L-NG-Nitroarginine methyl ester (10mg/kg) non-specific nitric oxide synthase inhibitor, 7-Nitroindazole (30mg/kg) specific neural nitric oxide synthase inhibitor, MK-801 (0.05mg/kg) an N-methyl-d-aspartate receptor antagonist but not aminoguanidine (50mg/kg) which is specific inducible nitric oxide synthase inhibitor as compared to the drugs when administered alone. No remarkable effect on locomotor activity was observed during open field test when the drugs were administered at the above mentioned doses. Therefore, it is evident that the antidepressant like effect of Dextromethorphan is owed due to its inhibitory effect on N-methyl-d-aspartate receptor and NO- Cyclic guanosine monophosphate pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  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. Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy.

    Science.gov (United States)

    Lane, M J; Thomas, G J

    1979-09-04

    Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in guanosine 5'-monophosphate (5'-rGMP) and guanosine 3':5'-monophosphate (cGMP) were determined as a function of temperature in the range 30-80 degrees C by means of laser-Raman spectroscopy. For each guanine nucleotide the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature: i.e., k psi = Ae-Ea/RT with A = 8.84 X 10(14) h-1 and Ea = 24.6 kcal/mol for 5'-rGMP and A = 3.33 X 10(13) h-1 and Ea = 22.2 kcal/mol for cGMP. Exchange of the 8-CH groups in guanine nucleotides is generally 2-3 times more rapid than in adenine nucleotides [cf. g. j. thomas, Jr., & J. Livramento (1975) Biochemistry 14, 5210-5218]. As in the case of adenine nucleotides, cyclic and 5' nucleotides of guanine exchange at markedly different rates at lower temperatures, with exchange in the cyclic nucleotide being the more facile. Each of the guanine nucleotides was prepared in four different isotopic modifications for Raman spectral analysis. The Raman frequency shifts resulting from the various isotopic substitutions have been tabulated, and assignments have been given for most of the observed vibrational frequencies.

  5. 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.

  6. 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.

  7. A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes.

    Science.gov (United States)

    Firsov, Alexander M; Rybalkina, Irina G; Kotova, Elena A; Rokitskaya, Tatyana I; Tashlitsky, Vadim N; Korshunova, Galina A; Rybalkin, Sergei D; Antonenko, Yuri N

    2018-02-01

    The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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...

  9. Involvement of NMDA receptors and L-arginine/nitric oxide/cyclic guanosine monophosphate pathway in the antidepressant-like effects of topiramate in mice forced swimming test.

    Science.gov (United States)

    Ostadhadi, Sattar; Khan, Muhammad Imran; Norouzi-Javidan, Abbas; Chamanara, Mohsen; Jazaeri, Farahnaz; Zolfaghari, Samira; Dehpour, Ahmad-Reza

    2016-04-01

    Topiramate (TPM) is an agent primarily used in the treatment of epilepsy. Using mice model of forced swimming test (FST) the current study was basically aimed to investigate the influence of TPM on depression by inhibiting NMDA receptor and nitric oxide-cGMP production. When TPM was administered in a dose of 20 and 30 mg/kg by i.p. route it reduced the immobility time during FST. However this effect of TPM (30 mg/kg, i.p.) in the FST was abolished when the mice were pretreated either with NMDA (75 mg/kg, i.p.), or l-arginine (750 mg/kg, i.p. NO precursor), or sildenafil (5mg/kg, i.p. Phosphodiesterase 5 inhibitor). The immobility time in the FST was reduced after administration of L-NAME (10mg/kg, i.p, a non-specific NOS inhibitor), 7-nitoinidazol (30 mg/kg, i.p. a nNOS inhibitor) or MK-801 (0.05 mg/kg, i.p, a NMDA receptor antagonist) in combination with a subeffective dose of TPM (10mg/kg, i.p.) as compared with single use of either drug. Co-administrated of lower doses of MK-801 (0.01 mg/kg) or L-NAME (1mg/kg) failed to effect immobility time. However, simultaneous administration of these two agents in the same doses with subeffective dose of TPM (10mg/kg, i.p.), reduced the immobility time during FST. None of these drugs were found to have a profound effect on the locomotor activity per se during the open field test. Taken together, our data demonstrates that TPM exhibit antidepressant-like effect which is accomplished either due to inhibition of NMDA receptors or NO-cGMP production. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Visualization of drug-nucleic acid interactions at atomic resolution. II. Structure of an ethidium/dinucleoside monophosphate crystalline complex, ethidium:5-iodocytidylyl(3'5')guanosine

    Energy Technology Data Exchange (ETDEWEB)

    Jain, S C; Tsair, C C; Sobell, H M

    1977-01-01

    Ethidium forms a second crystalline complex with the dinucleoside monophosphate 5-iodocytidyl(3'-5')guanosine (iodoCpG). These crystals are monoclinic, P2/sub 1/, with a = 14.06 A, b = 32.34 A, c = 16.53 A, ..beta.. = 117.8/sup 0/. The structure has been solved to atomic resolution using rigid-body Patterson vector search and Fourier methods, and refined by full matrix least-squares to a residual of 0.16 on 3180 observed reflections. Both iodoCpG molecules are hydrogen-bonded together by guanine.cytosine Watson--Crick base-pairing. Adjacent base-pairs within this paired iodoCpG structure and between neighboring iodoCpG molecules in adjoining unit cells are separated by 6.7 A. Approximate 2-fold symmetry is used in the interaction; this reflects the pseudo-2-fold symmetry axis of the phenanthridinium ring system in ethidium coinciding with the approximate 2-fold axis relating base-paired iodoCpG molecules. The phenyl and ethyl groups of the intercalated ethidium molecule lie in the narrow groove of the miniature iodoCpG double-helix. The stacked ethidium, however, lies in the opposite direction, its phenyl and ethyl groups neighboring iodine atoms on cytosine residues. Base-pairs within the paired nucleotide units are related by a twist of about 8/sup 0/. The magnitude of this angular twist reflects conformational changes in the sugar--phosphate chains accompanying intercalation. These primarily reflect the differences in ribose sugar ring puckering that are observed (i.e., both iodocytidine residues have C3' endo sugar conformations, while both guanosine residues have C2' endo sugar conformations), and alterations in the glycosidic torsional angles that describe the base-sugar orientation.

  11. Decreased levels of guanosine 3', 5'-monophosphate (cGMP) in cerebrospinal fluid (CSF) are associated with cognitive decline and amyloid pathology in Alzheimer's disease.

    Science.gov (United States)

    Ugarte, Ana; Gil-Bea, Francisco; García-Barroso, Carolina; Cedazo-Minguez, Ángel; Ramírez, M Javier; Franco, Rafael; García-Osta, Ana; Oyarzabal, Julen; Cuadrado-Tejedor, Mar

    2015-06-01

    Levels of the cyclic nucleotides guanosine 3', 5'-monophosphate (cGMP) or adenosine 3', 5'-monophosphate (cAMP) that play important roles in memory processes are not characterized in Alzheimer's disease (AD). The aim of this study was to analyse the levels of these nucleotides in cerebrospinal fluid (CSF) samples from patients diagnosed with clinical and prodromal stages of AD and study the expression level of the enzymes that hydrolyzed them [phosphodiesterases (PDEs)] in the brain of AD patients vs. For cGMP and cAMP CSF analysis, the cohort (n = 79) included cognitively normal participants (subjective cognitive impairment), individuals with stable mild cognitive impairment or AD converters (sMCI and cMCI), and mild AD patients. A high throughput liquid chromatography-tandem mass spectrometry method was used. Interactions between CSF cGMP or cAMP with mini-mental state examination (MMSE) score, CSF Aβ(1-42) and CSF p-tau were analysed. For PDE4, 5, 9 and 10 expression analysis, brains of AD patients vs. controls (n = 7 and n = 8) were used. cGMP, and not cAMP levels, were significantly lower in the CSF of patients diagnosed with mild AD when compared with nondemented controls. CSF levels of cGMP showed a significant association with MMSE-diagnosed clinical dementia and with CSF biomarker Aβ42 in AD patients. Significant increase in PDE5 expression was detected in temporal cortex of AD patients compared with that of age-matched healthy control subjects. No changes in the expression of others PDEs were detected. These results support the potential involvement of cGMP in the pathological and clinical development of AD. The cGMP reduction in early stages of AD might participate in the aggravation of amyloid pathology and cognitive decline. © 2014 British Neuropathological Society.

  12. Phase transformation of guanosine 5'-monophosphate in drowning-out crystallization: Comparison of experimental results with mathematical modeling

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Anh-Tuan; Kang, Jeong-Ki; Kim, Woo-Sik [Department of Chemical Engineering, Kyung Hee University, Seocheon-Dong, Giheung-Gu, 446-701 Yongin-Si (Korea, Republic of); Choi, Guang Jin [Department of Pharmaceutical Engineering, Inje University, 607 Uhbang-Dong, Gimhae, 621-746 Kyungnam (Korea, Republic of)

    2011-01-15

    The phase transformation of Guanosine 5{sup '}-Monophousphate (GMP) in drowning-out crystallization using a batch system was experimentally monitored and mathematically modeled. The solid (amorphous and crystalline GMP hydrate) and liquid phases of the GMP products were simultaneously monitored using a video microscope, FT-IR, and UV/Vis spectroscopy during the phase transformation. For the modeling, the phase transformation was assumed to occur via the simultaneous dissolution of amorphous GMP and growth of crystalline GMP hydrate in the solution. Based on a comparison of the experimental results and model predictions, both the dissolution and growth of the GMP solids were found to contribute competitively to the phase transformation. When varying the crystallization conditions, in this case the agitation speed and feed concentration, the phase transformation was significantly promoted when increasing the agitation speed, yet independent of the feed concentration. The simple mathematical model used for the GMP phase transformation was quite successful in describing the experimental results. (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  13. The Role of Growth Factors (VEGF, TGF-β1 and Cyclic Guanosine Monophosphate in the Formation of Pulmonary Hypertension in Children with Bronchopulmonary Dysplasia

    Directory of Open Access Journals (Sweden)

    A.S. Senatorova

    2013-10-01

    Full Text Available In 82 children with bronchopulmonary dysplasia (from 1 to 36 months of corrected age we investigated the level of VEGF, TGF-β1 in blood and cyclic guanosine monophosphate (cGMP in sputum. It was revealed that children with bronchopulmonary dysplasia had a significant increase in TGF-β1 (p < 0.05 and cGMP (p < 0.01–0.001, reduced VEGF (p < 0.05, indicating inhibition of angiogenesis, activation of fibrosis factors and endothelium-dependent vasodilation. Reliable direct dependence of activation of TGF-β1 in blood and cGMP in sputum, as well as inverse correlation between VEGF in blood and rLA had been proved, which gave reason to think of pulmonary hypertension as an adverse factor in fibrosis activation and angiogenesis inhibition in children with bronchopulmonary dysplasia. Reduced oxygen saturation and oxygen partial pressure moderately activated cGMP, but did not provide a sufficient reduction of pressure in the pulmonary artery.

  14. Host-guest chemistry of dendrimer-drug complexes. 4. An in-depth look into the binding/encapsulation of guanosine monophosphate by dendrimers.

    Science.gov (United States)

    Hu, Jingjing; Fang, Min; Cheng, Yiyun; Zhang, Jiahai; Wu, Qinglin; Xu, Tongwen

    2010-06-03

    In the present study, we investigated the host-guest chemistry of dendrimer/guanosine monophosphate (GMP) and present an in-depth look into the binding/encapsulation of GMP by dendrimers using NMR studies. (1)H NMR spectra showed a significant downfield shift of methylene protons in the outmost layer of the G5 dendrimer, indicating the formation of ion pairs between cationic amine groups of dendrimer and anionic phosphate groups of GMP. Chemical shift titration results showed that the binding constant between G5 dendrimer and GMP is 17,400 M(-1) and each G5 dendrimer has 107 binding sites. The binding of GMP to dendrimers prevents its aggregation in aqueous solutions and thereby enhances its stability. Nuclear Overhauser effect measurements indicated that a GMP binding and encapsulation balance occurs on the surface and in the interior of dendrimer. The binding/encapsulation transitions can be easily tailored by altering the surface and interior charge densities of the dendrimer. All these findings provide a new insight into the host-guest chemistry of dendrimer/guest complexes and may play important roles in the study of dendrimer/DNA aggregates by a "bottom-up" strategy.

  15. Effects of dietary administration of guanosine monophosphate on the growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major.

    Science.gov (United States)

    Hossain, Md Sakhawat; Koshio, Shunsuke; Ishikawa, Manabu; Yokoyama, Saichiro; Sony, Nadia Mahjabin

    2016-10-01

    The present study explored the dietary administration effects of guanosine monophosphate (GMP) on growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major. A semi-purified basal diet supplemented with 0% (Control), 0.1% (GMP-0.1), 0.2% (GMP-0.2), 0.4% (GMP-0.4) and 0.8% (GMP-0.8) purified GMP to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4 g) for 56 days. The obtained results clearly indicated that, growth performance of red sea bream enhanced by dietary GMP supplementation compared to control and significantly higher final weight was found in fish fed diet group GMP-0.4. Specific growth rate (SGR) and percent weight gain (%WG) also significantly higher in diet group GMP-0.4 in compared to control and it was not differed (P > 0.05) with diet group GMP-0.8. Feed intake significantly increased with the supplementation of GMP. Feed conversion efficiency (FCE) and protein efficiency ratio (PER) also improved (P  0.05) by GMP supplementation. In contrast, catalase activity decreased with GMP supplementation. In terms of oxidative stress GMP-0.2 showed best condition with low oxidative stress and high antioxidant level. Moreover, the fish fed GMP supplemented diets had better improvement (P red sea bream, which is also in line with the most of the growth performance and health parameters of the fish. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Visualization of drug-nucleic acid interactions at atomic resolution. VIII. Structures of two ethidium/dinucleoside monophosphate crystalline complexes containing ethidium: cytidylyl(3'-5') guanosine

    International Nuclear Information System (INIS)

    Jain, S.C.; Sobell, H.M.

    1984-01-01

    This paper describes two complexes containing ethidium and the dinucleoside monophosphate, cytidylyl(3'-5')guanosine (CpG). Both crystals are monoclinic, space group P2 1 , with unit cell dimensions as follows: modification 1: a = 13.64 A, b = 32.16 A, c = 14.93 A, β = 114.8 0 and modification 2: a = 13.79 A, b = 31.94 A, c = 15.66 A, β = 117.5 0 . Each structure has been solved to atomic resolution and refined by Fourier and least squares methods; the first has been refined to a residual of 0.187 on 1903 reflections, while the second has been refined to a residual of 0.187 on 1001 reflections. The asymmetric unit in both structures contains two ethidium molecules and two CpG molecules; the first structure has 30 water molecules (a total of 158 non-hydrogen atoms), while the second structure has 19 water molecules (a total of 147 non-hydrogen atoms). Both structures demonstrate intercalation of ethidium between base-paired CpG dimers. In addition, ethidium molecules stack on either side of the intercalated duplex, being related by a unit cell translation along the a axis. The basic feature of the sugar-phosphate chains accompanying ethidium intercalation in both structures is: C3' endo (3'-5') C2' endo. This mixed sugar-puckering pattern has been observed in all previous studies of ethidium intercalation and is a feature common to other drug-nucleic acid structural studies carried out in the authors laboratory. The authors discussed this further in this paper and in the accompanying papers

  17. Studies on Relationship between Serum Nitric Oxide and Plasma Cyclic Guanosine Monophosphate and Prolonged Bleeding after Medical Abortion as well as Prophylaxis and Treatment of Bleeding with Traditional Chinese Medicine

    Institute of Scientific and Technical Information of China (English)

    廖玎玲; 谭布珍; 辛华; 贺晓菊

    1999-01-01

    Objectives To study the relationship between serum nitric oxide(NO and plasma cyclic guanosine monophosphate(cGMP)and prolonged bleeding after medical abortion.Methods A total of 120women having received medical abortions at random were recruited and divided into two groups:the one(Group A,n=60) taking “Gong-Fu Mixture(Uterus-Recovering Mixture)”and the other(Group B,n=60)not taking it after abortion.On d 10,20 and 30 after medical abortion,serum NO and plasma cGMP were tested before and after mifepristone administration and 10 d later by Gresis reac-tion method and radioimmunoassay respectively.Results NO concentration in serum and cGMP concentration in plasma decreased signifi-cantly after taking mifeprlstone given(P<0. 05).Ten days later,the number of thos ewith bleeding discontinuation in the group A was significantly greater than that in the group B(P<0.05).Serum NO level and plasma cGMP level in the group A de-creased more significantly than those in the group B(P<0. 05).Conclusion The slow decrease of serum NO and plasma cGMP is closely related to prolonged bleeding after medical abortion.“Gong-Fu Mixture(uterus-recovering mixture)”is effective in prevention and treatment of prolonged bleeding.

  18. Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.

    Science.gov (United States)

    Zhang, Yanhua; Dufield, Dawn; Klover, Jon; Li, Wenlin; Szekely-Klepser, Gabriella; Lepsy, Christopher; Sadagopan, Nalini

    2009-02-15

    An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.

  19. Cyclic guanosine monophosphate in the regulation of the cell function

    Directory of Open Access Journals (Sweden)

    Małgorzata Zbrojkiewicz

    2016-12-01

    Full Text Available Intracellular concentration of cGMP depends on the activity of guanylate cyclase, responsible for its synthesis, on the activity of cyclic nucleotide degrading enzymes - phosphodiesterases (PDEs. There are two forms of guanylate cyclase: the membrane-bound cyclase and the soluble form. The physiological activators of the membrane guanylate cyclase are natriuretic peptides (NPs, and of the cytosolic guanylate cyclase - nitric oxide (NO and carbon monoxide (CO. Intracellular cGMP signaling pathways arise from its direct effect on the activity of G protein kinases, phosphodiesterases and cyclic nucleotide dependent cation channels. It has been shown in recent years that cGMP can also affect other signal pathways in cell signaling activity involving Wnt proteins and sex hormones. The increased interest in the research on the role of cGMP, resulted also in the discovery of its role in the regulation of phototransduction in the eye, neurotransmission, calcium homeostasis, platelet aggregation, heartbeat, bone remodeling, lipid metabolism and the activity of the cation channels. Better understanding of the mechanisms of action of cGMP in the regulation of cell function can create new opportunities for the cGMP affecting drugs use in the pharmacotherapy.

  20. Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

    Science.gov (United States)

    Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

    2012-03-01

    Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

  1. 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.

  2. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A; Turek, Ilona S.

    2017-01-01

    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.

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

    KAUST Repository

    Parrott, Brian

    2011-01-01

    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

  4. Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

    International Nuclear Information System (INIS)

    Hamel, E.; Batra, J.K.; Lin, C.M.

    1986-01-01

    Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

  5. Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1989-01-01

    Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated b...... a purF, a purL or a purM mutation. A revised map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE....

  6. Inhibition of translation by 7-methyl guanosine (m7G) nucleotide cap analogs with derivatized 5'-monophosphates

    International Nuclear Information System (INIS)

    Tahara, S.M.; Darzynkiewicz, E.; Ekiel, I.

    1986-01-01

    Recognition of the 5'-m 7 GpppN (cap) structure of eukaryote mRNA is an important step of translation initiation as shown by the potent inhibitory effect of m 7 G nucleotides on this process. A comparison of cap analogs as competitive inhibitors of initiation has allowed the authors to map probable protein-ligand contact points between the cap and cognate cap binding proteins (CBPs). Recently, several new derivatives of m 7 GMP (1) with modified phosphates were synthesized: m 7 G 5'-phosphite (2), m 7 G 5'-phosphoramidate (3), m 7 G 5'-methylphosphonate (4), and m 7 G 5'-phosphate-O-methyl ester (5). In addition, 7,8-dimethyl GMP (6) and 7-methyl 8-amino GMP (7) were synthesized. 6 and7 are primarily syn and anti respectively, relative to the glycosidic bond as shown by solution NMR studies. Inhibition by analogs on total translation in reticulocyte lysate and binding of mRNA to rabbit reticulocyte ribosomes was found to be: 1 = 3 > 5 > 4 > 2. The inhibitory activity of 3 was unexpected since it is isosteric with 4, however it suggested that electron configuration and/or the ability to form a hydrogen bond between protein and the phosphate moiety might be important for ligand binding. 7 was more inhibitory than 6. The latter two are isosteric therefore differences in electron delocalization and/or syn-anti conformation are likely to be the reason(s) for the observed difference

  7. Discovery of inhibitors of Leishmania β-1,2-mannosyltransferases using a click-chemistry-derived guanosine monophosphate library.

    Directory of Open Access Journals (Sweden)

    Phillip van der Peet

    Full Text Available Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose-dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-D-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism.

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

    Science.gov (United States)

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

    2016-05-01

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

  9. 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.

  10. Hierarchical Formation of Fibrillar and Lamellar Self-Assemblies from Guanosine-Based Motifs

    Directory of Open Access Journals (Sweden)

    Paolo Neviani

    2010-01-01

    Full Text Available Here we investigate the supramolecular polymerizations of two lipophilic guanosine derivatives in chloroform by light scattering technique and TEM experiments. The obtained data reveal the presence of several levels of organization due to the hierarchical self-assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures. The elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties.

  11. Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents.

    Science.gov (United States)

    Röhrig, Teresa; Pacjuk, Olga; Hernández-Huguet, Silvia; Körner, Johanna; Scherer, Katharina; Richling, Elke

    2017-11-04

    Background: Phosphodiesterases (PDEs) play a major role in the regulation of cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts ( Arbutus unedo , Camellia sinensis , Cynara scolymus , Zingiber officinale ) with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit ( Arbutus unedo ) and tea ( Camellia sinensis ) extracts did not inhibit PDE markedly. Alternatively, artichoke ( Cynara scolymus ) extract had a significant inhibitory influence on PDE activity (IC 50 = 0.9 ± 0.1 mg/mL) as well as its flavone luteolin (IC 50 = 41 ± 10 μM) and 3,4-dicaffeoylquinic acid (IC 50 > 1.0 mM). Additionally, the ginger ( Zingiber officinale ) extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC 50 = 1.7 ± 0.2 mg/mL and IC 50 > 1.7 mM, respectively). Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC 50 = 455 ± 19 μg/mL). Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research.

  12. Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents

    Directory of Open Access Journals (Sweden)

    Teresa Röhrig

    2017-11-01

    Full Text Available Background: Phosphodiesterases (PDEs play a major role in the regulation of cyclic adenosine monophosphate (cAMP- and cyclic guanosine monophosphate (cGMP-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts (Arbutus unedo, Camellia sinensis, Cynara scolymus, Zingiber officinale with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit (Arbutus unedo and tea (Camellia sinensis extracts did not inhibit PDE markedly. Alternatively, artichoke (Cynara scolymus extract had a significant inhibitory influence on PDE activity (IC50 = 0.9 ± 0.1 mg/mL as well as its flavone luteolin (IC50 = 41 ± 10 μM and 3,4-dicaffeoylquinic acid (IC50 > 1.0 mM. Additionally, the ginger (Zingiber officinale extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC50 = 1.7 ± 0.2 mg/mL and IC50 > 1.7 mM, respectively. Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC50 = 455 ± 19 μg/mL. Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research.

  13. 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.

  14. Acridone-based inhibitors of inosine 5'-monophosphate dehydrogenase: discovery and SAR leading to the identification of N-(2-(6-(4-ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2- fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (BMS-566419).

    Science.gov (United States)

    Watterson, Scott H; Chen, Ping; Zhao, Yufen; Gu, Henry H; Dhar, T G Murali; Xiao, Zili; Ballentine, Shelley K; Shen, Zhongqi; Fleener, Catherine A; Rouleau, Katherine A; Obermeier, Mary; Yang, Zheng; McIntyre, Kim W; Shuster, David J; Witmer, Mark; Dambach, Donna; Chao, Sam; Mathur, Arvind; Chen, Bang-Chi; Barrish, Joel C; Robl, Jeffrey A; Townsend, Robert; Iwanowicz, Edwin J

    2007-07-26

    Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5'-monophosphate to xanthosine-5'-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model.

  15. The immediate nucleotide precursor, guanosine triphosphate, in the riboflavin biosynthetic pathway

    International Nuclear Information System (INIS)

    Mitsuda, Hisateru; Nakajima, Kenji; Nadamoto, Tomonori

    1977-01-01

    In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashbyii. The added purines, at 10 -4 M, were effectively incorporated into riboflavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the course of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed. (auth.)

  16. Ag(I)-mediated homo and hetero pairs of guanosine and cytidine: monitoring by circular dichroism spectroscopy.

    Science.gov (United States)

    Goncharova, Iryna

    2014-01-24

    Ag(I)-containing compounds are attractive as antibacterial and antifungal agents. The renewed interest in the application of silver(I) compounds has led to the need for detailed knowledge of the mechanism of their action. One of the possible ways is the coordination of Ag(I) to G-C pairs of DNA, where Ag(+) ions form Ag(I)-mediated base pairs and inhibit the transcription. Herein, a systematic chiroptical study on silver(I)-mediated homo and mixed pairs of the C-G complementary-base derivatives cytidine(C) and 5'-guanosine monophosphate(G) in water is presented. Ag(I)-mediated homo and hetero pairs of G and C and their self-assembled species were studied under two pH levels (7.0 and 10.0) by vibrational (VCD) and electronic circular dichroism(ECD). VCD was used for the first time in this field and showed itself to be a powerful method for obtaining specific structural information in solution. Based on results of the VCD experiments, the different geometries of the homo pairs were proposed under pH 7.0 and 10.0. ECD was used as a diagnostic tool to characterize the studied systems and as a contact point between the previously defined structures of the metal or proton mediated pairs of nucleobases and the systems studied here. On the basis of the obtained data, the formation of the self-assembled species of cytidine with a structure similar to the i-motif structure in DNA was proposed at pH 10.0. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Pitts, William J; Guo, Junqing; Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Watterson, Scott H; Bednarz, Mark S; Chen, Bang Chi; Barrish, Joel C; Bassolino, Donna; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2002-08-19

    A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

  18. Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase.

    Science.gov (United States)

    Watterson, Scott H; Liu, Chunjian; Dhar, T G Murali; Gu, Henry H; Pitts, William J; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2002-10-21

    A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

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

    International Nuclear Information System (INIS)

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang; Lu, Weiqiang; Huang, Jin

    2016-01-01

    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_5_0 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.

  20. 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.

  1. Cyclic guanosine monophosphate does not inhibit gonadotropin-induced activation of mitogen-activated protein kinase 3/1 in pig cumulus-oocyte complexes

    Czech Academy of Sciences Publication Activity Database

    Blaha, Milan; Němcová, Lucie; Procházka, Radek

    2015-01-01

    Roč. 13, č. 1 (2015) ISSN 1477-7827 R&D Projects: GA ČR GAP502/11/0593; GA MZe QI101A166 Institutional support: RVO:67985904 Keywords : pig * oocyte * cGMP Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.147, year: 2015

  2. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

    DEFF Research Database (Denmark)

    Adari, H; Lowy, D R; Willumsen, B M

    1988-01-01

    A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as w...

  3. Structural characterization of the stringent response related exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family

    DEFF Research Database (Denmark)

    Kristensen, Ole; Laurberg, Martin; Liljas, Anders

    2004-01-01

    Exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes play central roles in the bacterial stringent response induced by starvation. The high-resolution crystal structure of the putative Aquifex aeolicus PPX/GPPA phosphatase from the actin-like ATPase domain superfamily has...

  4. Guanosine radical reactivity explored by pulse radiolysis coupled with transient electrochemistry.

    Science.gov (United States)

    Latus, A; Alam, M S; Mostafavi, M; Marignier, J-L; Maisonhaute, E

    2015-06-04

    We follow the reactivity of a guanosine radical created by a radiolytic electron pulse both by spectroscopic and electrochemical methods. This original approach allows us to demonstrate that there is a competition between oxidation and reduction of these intermediates, an important result to further analyse the degradation or repair pathways of DNA bases.

  5. Charge-density-wave instabilities expected in monophosphate tungsten bronzes

    International Nuclear Information System (INIS)

    Canadell, E.; Whangbo, M.

    1991-01-01

    On the basis of tight-binding band calculations, we examined the electronic structures of the tungsten oxide layers found in the monophosphate tungsten bronze (MPTB) phases. The Fermi surfaces of these MPTB phases consist of five well-nested one- and two-dimensional pieces. We calculated the nesting vectors of these Fermi surfaces and discussed the expected charge-density-wave instabilities

  6. Metabolism of inositol 4-monophosphate in rat mammalian tissues

    International Nuclear Information System (INIS)

    Delvaux, A.; Dumont, J.E.; Erneux, C.

    1987-01-01

    Rat brain soluble fraction contains an enzymatic activity that dephosphorylates inositol 1,4-bisphosphate (Ins(1,4)P2). We have used anion exchange h.p.l.c. in order to identify the inositol monophosphate product of Ins(1,4)P2 hydrolysis (i.e. Ins(1)P1, Ins(4)P1 or both). When [ 3 H]Ins(1,4)P2 was used as substrate, we obtained an inositol monophosphate isomer that was separated from the co-injected standard [ 3 H]Ins(1)P1. This suggested an Ins(1,4)P21-phosphatase pathway leading to the production of the inositol 4-monophosphate isomer. The dephosphorylation of [ 32 P]Ins(4)P1 was measured in rat brain, liver and heart soluble fraction and was Li+-sensitive. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved a monophosphate phosphatase activity that hydrolyzed both [ 3 H]Ins(1)P1 and [4- 32 P]Ins(4)P1 isomers

  7. Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

    2002-10-21

    A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

  8. Structure of 7,12-dimethylbenz(a)anthracene-guanosine adducts.

    Science.gov (United States)

    Jeffrey, A M; Blobstein, S H; Weinstein, I B; Beland, F A; Harvey, R G; Kasai, H; Nakanishi, K

    1976-07-01

    Arene oxides have been proposed as the reactive intermediates in the process of carcinogenesis induced by polycyclic aromatic hydrocarbons. The present study defines the structures of four guanosine adducts formed by the reaction of 7,12-dimethylbenz[a]anthracene-5,6-oxide with polyguanylic acid. The modified polymer was hydrolyzed to nucleotides and the hydrophobic guanosine adducts separated from unmodified guanosine by LH-20 column chromatograhy. The adducts were further resolved into four components (I-IV) by reverse phase high pressure liquid chromatography. Analysis of the ultraviolet, circular dichroism, mass, and proton magnetic resonance spectra of these compounds, or their acetate and free base derivatives, indicates that in all four compounds the aromatic hydrocarbon is present on the 2 amino group of guanine. Compounds I and IV, and II and III constitute diastereoisomeric pairs, respectively. In the I and IV pair, the adducts result from addition at the 6 position of the original dimethylbenz[a]anthracene oxide, whereas in the II and III pair, the addition occurs at the 5 position. Indirect evidence suggests that trans opening of the oxide occurred in all cases but this remains to be established.

  9. Structure of 7,12-dimethylbenz(a)anthracene-guanosine adducts.

    Science.gov (United States)

    Jeffrey, A M; Blobstein, S H; Weinstein, I B; Beland, F A; Harvey, R G; Kasai, H; Nakanishi, K

    1976-01-01

    Arene oxides have been proposed as the reactive intermediates in the process of carcinogenesis induced by polycyclic aromatic hydrocarbons. The present study defines the structures of four guanosine adducts formed by the reaction of 7,12-dimethylbenz[a]anthracene-5,6-oxide with polyguanylic acid. The modified polymer was hydrolyzed to nucleotides and the hydrophobic guanosine adducts separated from unmodified guanosine by LH-20 column chromatograhy. The adducts were further resolved into four components (I-IV) by reverse phase high pressure liquid chromatography. Analysis of the ultraviolet, circular dichroism, mass, and proton magnetic resonance spectra of these compounds, or their acetate and free base derivatives, indicates that in all four compounds the aromatic hydrocarbon is present on the 2 amino group of guanine. Compounds I and IV, and II and III constitute diastereoisomeric pairs, respectively. In the I and IV pair, the adducts result from addition at the 6 position of the original dimethylbenz[a]anthracene oxide, whereas in the II and III pair, the addition occurs at the 5 position. Indirect evidence suggests that trans opening of the oxide occurred in all cases but this remains to be established. PMID:821053

  10. m/sup 7/Guanosine in tRNA of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Shugart, L; Chastain, B

    1979-01-01

    The nucleoside content of six different individual tRNAs was re-investigated. Particular emphasis was placed on obtaining an accurate, quantitative estimation of the amount of the minor nucleoside m/sup 7/guanosine which was found to occur at less than one full residue in tRNA/sup Phe/(E.coli) and tRNA/sup Arg/(E. coli) but not in the other species of tRNAs examined. Several hypotheses to explain this observation are discussed.

  11. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

    OpenAIRE

    Garone, Caterina; Garc??a-D??az, Beatriz; Emmanuele, Valentina; L??pez Garc??a, Luis Carlos; Tadesse, Saba; Akman, Hasan O.; Tanji, Kurenai; Quinzii, Catarina M.; Hirano, Michio

    2014-01-01

    Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 −/− ) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-...

  12. Guanosine prevents behavioral alterations in the forced swimming test and hippocampal oxidative damage induced by acute restraint stress.

    Science.gov (United States)

    Bettio, Luis E B; Freitas, Andiara E; Neis, Vivian B; Santos, Danúbia B; Ribeiro, Camille M; Rosa, Priscila B; Farina, Marcelo; Rodrigues, Ana Lúcia S

    2014-12-01

    Guanosine is a guanine-based purine that modulates glutamate uptake and exerts neurotrophic and neuroprotective effects. In a previous study, our group demonstrated that this endogenous nucleoside displays antidepressant-like properties in a predictive animal model. Based on the role of oxidative stress in modulating depressive disorders as well as on the association between the neuroprotective and antioxidant properties of guanosine, here we investigated if its antidepressant-like effect is accompanied by a modulation of hippocampal oxidant/antioxidant parameters. Adult Swiss mice were submitted to an acute restraint stress protocol, which is known to cause behavioral changes that are associated with neuronal oxidative damage. Animals submitted to ARS exhibited an increased immobility time in the forced swimming test (FST) and the administration of guanosine (5mg/kg, p.o.) or fluoxetine (10mg/kg, p.o., positive control) before the exposure to stressor prevented this alteration. Moreover, the significantly increased levels of hippocampal malondialdehyde (MDA; an indicator of lipid peroxidation), induced by ARS were not observed in stressed mice treated with guanosine. Although no changes were found in the hippocampal levels of reduced glutathione (GSH), the group submitted to ARS procedure presented enhanced glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) activities and reduced catalase (CAT) activity in the hippocampus. Guanosine was able to prevent the alterations in GPx, GR, CAT activities, and in SOD/CAT activity ratio, but potentiated the increase in SOD activity elicited by ARS. Altogether, the present findings indicate that the observed antidepressant-like effects of guanosine might be related, at least in part, to its capability of modulating antioxidant defenses and mitigating hippocampal oxidative damage induced by ARS. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Inosine monophosphate dehydrogenase messenger RNA expression is correlated to clinical outcomes in mycophenolate mofetil-treated kidney transplant patients, whereas inosine monophosphate dehydrogenase activity is not

    NARCIS (Netherlands)

    Sombogaard, Ferdi; Peeters, Annemiek M. A.; Baan, Carla C.; Mathot, Ron A. A.; Quaedackers, Monique E.; Vulto, Arnold G.; Weimar, Willem; van Gelder, Teun

    2009-01-01

    Measurement of the pharmacodynamic biomarker inosine monophosphate dehydrogenase (IMPDH) activity in renal transplant recipients has been proposed to reflect the biological effect better than using pharmacokinetic parameters to monitor mycophenolate mofetil therapy. The IMPDH assays are however

  14. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

    Science.gov (United States)

    Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

    2004-01-01

    The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

  15. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    Science.gov (United States)

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. 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.

  17. Monophosphate end groups produced in radiation induced strand breakage in DNA

    International Nuclear Information System (INIS)

    Kay, E.; Ward, J.F.

    1976-01-01

    A solution of DNA was gamma-irradiated and treated with monophosphatase for studies on the amount of inorganic phosphate released as a function of time. Studies were also conducted on: effect of alkali on yield of monophosphate end groups; induction of DNA strand breaks by treatment with DNAase; initial G values for monophosphate termini; and effect of alkali on radioinduced DNA damage

  18. A simplified radioimmunoassay of adenosine-3':5'-monophosphate

    International Nuclear Information System (INIS)

    Katoh, Yoshiki; Takezawa, Junichi; Suzuki, Morio; Kuninaka, Akira; Yoshino, Hiroshi

    1975-01-01

    Dextran-coated charcoal was proved to be able to separate free adenosine-3':5'monophosphate (cAMP) from antibody-bound cAMP. Only free cAMO was adsorbed on dextran-coated charcoal within 1 min after contacting the charcoal. In a reaction mixture of cAMP and anti-cAMP-plasma, most of antibody-bound cAMP had not been adsorbed 4 min after contacting. The data obtained were found to be almost the same as the data of another experiment using cellulose ester filter separation technique. Thus, dextran-coated charcoal could be employed to simplify the radioimmunoassay of cAMP. (author)

  19. Adenosine, but not guanosine, protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity.

    Science.gov (United States)

    Menezes, Camila Braz; Frasson, Amanda Piccoli; Meirelles, Lucia Collares; Tasca, Tiana

    2017-02-01

    Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5'-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd2+ Chemical Stimulus.

    Science.gov (United States)

    Lunn, Samantha M L; Hribesh, Samira; Whitfield, Colette J; Hall, Michael J; Houlton, Andrew; Bronowska, Agnieszka K; Tuite, Eimer M; Pike, Andrew R

    2018-03-25

    The on-column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio-directed Cd 2+ coordination; a route for healing DNA damage. Short oligomers are G-specifically thiolated through a modified on-column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd 2+ ions to a duplex containing a highly disrupted tG central mismatch sequence, 3'-A 6 tG 4 T 6 -5', suggests a (tG) 8 Cd 2 central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal-induced healing of the thiolated duplex. The 2 nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd 2+ ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Cyclic adenosine 3:5-monophosphate binding proteins in Hartmannella culbertsoni

    International Nuclear Information System (INIS)

    Verma, A.K.; Krishna Murti, C.R.

    1976-01-01

    When 100, 000 g supernatant fractions of homogenates of Hartmannella culbertsoni were incubated with ('- 3 H)-cyclic adenosine 3 : 5 monophosphate and passed through a sephadex G-100 column, radioactivity appeared with protein fractions eluted after the void colume. About 75% radioactivity bound to these fractions was recovered as cyclic adenosine 3 : 5 monophosphate. Unlabelled cAMP diluted the amount of radioactivity bound. Adenosine, deoxyadenosine, 5-AMP, 3-AMP, ADP and ATP did not inhibit binding. (author)

  2. Method of preparing tritium-labelled thymidine-5'-monophosphates of high specific activity

    International Nuclear Information System (INIS)

    Filip, J.; Vesely, J.; Cihak, A.

    1976-01-01

    A method is described of preparing thymidine-5'-monophosphates labelled with tritium of high specific activity based on enzyme synthesis in vitro. Phosphorylation was carried out using the catalytic effect of an enzyme contained in the supernatant fraction prepared from Yoshida ascites carcinoma in rats. The course of the enzyme reaction can be controlled by the concentration of the individual reaction mixture components. The method described allows obtaining thymidine-5'-monophosphate of radiochemical purity better than 95%. (J.B.)

  3. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency.

    Science.gov (United States)

    Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

    2014-08-01

    Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2(-/-)) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2(-/-) mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2(-/-200dCMP/) (dTMP)) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  4. The fate of H atom adducts to 3'-uridine monophosphate.

    Science.gov (United States)

    Wang, Ran; Zhang, Ru Bo; Eriksson, Leif A

    2010-07-29

    The stabilities of the adducts deriving from H free radical addition to the O2, O4, and C5 positions of 3'-uridine monophosphate (3'UMP) are studied by the hybrid density functional B3LYP approach. Upon H atom addition at the O2 position, a concerted low-barrier proton-transfer process will initially occur, followed by the potential ruptures of the N-glycosidic or beta-phosphate bonds. The rupture barriers are strongly influenced by the rotational configuration of the phosphate group at the 3' terminal, and are influenced by bulk solvation effects. The O4-H adduct has the highest thermal stability, as the localization of the unpaired electron does not enable cleavage of either the C1'-N1 or the C3'-O(P) bonds. For the most stable adduct, with H atom added to the C5 position, the rate-controlled step is the H2'a abstraction by the C6 radical site, after which the subsequent strand rupture reactions proceed with low barriers. The main unpaired electron densities are presented for the transient species. Combined with previous results, it is concluded that the H atom adducts are more facile to drive the strand scission rather than N-glycosidic bond ruptures within the nucleic acid bases.

  5. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    International Nuclear Information System (INIS)

    Chen, B.X.; Hubbard, K.; Ide, H.; Wallace, S.S.; Erlanger, B.F.

    1990-01-01

    A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions

  6. Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

    Science.gov (United States)

    Pavan Kumar, Y; Saha, Puja; Saha, Dhurjhoti; Bessi, Irene; Schwalbe, Harald; Chowdhury, Shantanu; Dash, Jyotirmayee

    2016-03-02

    The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.

    OpenAIRE

    Wong, S K; Martin, B R; Tolkovsky, A M

    1985-01-01

    We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guano...

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

    KAUST Repository

    Rhamani, Saher; Chaix, Arnaud; Aggad, Dina; Hoang, Phuong Mai; Moosa, Basem; Garcia, Marcel; Gary-Bobo, Magali; Charnay, Clarence; Almalik, Abdulaziz; Durand, Jean-Olivier; Khashab, Niveen M.

    2017-01-01

    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.

  9. 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.

  10. Short-term treatment with budesonide does not improve hyperresponsiveness to adenosine 5 '-monophosphate in COPD

    NARCIS (Netherlands)

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

    The role of inhaled corticosteroids in the treatment of chronic obstructive pulmonary disease (COPD) is unclear. We investigated the effects of budesonide on airway hyperresponsiveness (AHR) to methacholine (MCh) and adenosine 5'-monophosphate (AMP), to which we hypothesized the existence of greater

  11. 3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

    Science.gov (United States)

    Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2003-10-20

    A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

  12. A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase.

    Science.gov (United States)

    Dhar, T G Murali; Liu, Chunjian; Pitts, William J; Guo, Junquing; Watterson, Scott H; Gu, Henry; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Barrish, Joel C; Hollenbaugh, Diane; Iwanowicz, Edwin J

    2002-11-04

    A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.

  13. Adenosine 5'-Monophosphate Aerosol Challenge Does Not Provoke Airflow Limitation in Healthy Cats

    Directory of Open Access Journals (Sweden)

    K. Vondráková

    2006-01-01

    Full Text Available The purpose of our study was to investigate the effects of nebulized adenosine 5'- monophosphate on airflow limitation in healthy cats determined by barometric whole body plethysmography (BWBP, in comparison to the effects of carbachol. Ten healthy 4- to 6-year-old domestic shorthair cats were included in the study. Each cat was placed in a BWBP plexiglass chamber (volume 38 l. Changes in box pressure were measured at baseline and after nebulization of vehicle and increasing concentrations of carbachol and adenosine 5'- monophosphate. Airway responsiveness was monitored as increases in enhanced pause (PENH, a unitless variable derived from dose-response curves estimating airflow limitation. The chosen endpoint was the agonist concentration which increased PENH to 300% of the value obtained after saline nebulization (PCPENH 300. Inter-day repeatability of measurements was assessed by repeated bronchoprovocations with both agonists 2-3 days apart. For carbachol, PCPENH300 was reached in all cats and correlated significantly between days (mean ± SD; 0.54 ± 0.42 mg/ml and 0.64 ± 0.45 mg/ml respectively; r = 0.58, p < 0.05 In contrast, we found no reaction to adenosine 5'- monophosphate even with the highest concentration nebulized during both measurements. At baseline, mean ± SD PENH was 0.47 ± 0.18 and 0.58 ± 0.24 (measurements 1 and 2, whereas PENH after 500 mg/ml adenosine 5'- monophosphate was 0.46 ± 0.20 and 0.71 ± 0.37. All bronchoprovocation tests were well tolerated by the cats. We conclude that healthy airways in cats do not demonstrate airway responsiveness to inhaled adenosine 5'- monophosphate. This is in agreement with observations in humans as well as our previous findings in dogs, where adenosine 5'- monophosphate had no effect on healthy canine airways, but caused significant airflow limitation after induction of acute bronchitis. To define the value of bronchoprovocation testing with adenosine 5'- monophosphate in the feline

  14. Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine.

    Science.gov (United States)

    Li, Yang; Zhu, Xujun; Zhang, Xueyu; Fu, Jing; Wang, Zhiwen; Chen, Tao; Zhao, Xueming

    2016-06-03

    Genome streamlining has emerged as an effective strategy to boost the production efficiency of bio-based products. Many efforts have been made to construct desirable chassis cells by reducing the genome size of microbes. It has been reported that the genome-reduced Bacillus subtilis strain MBG874 showed clear advantages for the production of several heterologous enzymes including alkaline cellulase and protease. In addition to enzymes, B. subtilis is also used for the production of chemicals. To our best knowledge, it is still unknown whether genome reduction could be used to optimize the production of chemicals such as nucleoside products. In this study, we constructed a series of genome-reduced strains by deleting non-essential regions in the chromosome of B. subtilis 168. These strains with genome reductions ranging in size from 581.9 to 814.4 kb displayed markedly decreased growth rates, sporulation ratios, transformation efficiencies and maintenance coefficients, as well as increased cell yields. We re-engineered the genome-reduced strains to produce guanosine and thymidine, respectively. The strain BSK814G2, in which purA was knocked out, and prs, purF and guaB were co-overexpressed, produced 115.2 mg/L of guanosine, which was 4.4-fold higher compared to the control strain constructed by introducing the same gene modifications into the parental strain. We also constructed a thymidine producer by deleting the tdk gene and overexpressing the prs, ushA, thyA, dut, and ndk genes from Escherichia coli in strain BSK756, and the resulting strain BSK756T3 accumulated 151.2 mg/L thymidine, showing a 5.2-fold increase compared to the corresponding control strain. Genome-scale genetic manipulation has a variety of effects on the physiological characteristics and cell metabolism of B. subtilis. By introducing specific gene modifications related to guanosine and thymidine accumulation, respectively, we demonstrated that genome-reduced strains had greatly improved

  15. Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli

    DEFF Research Database (Denmark)

    Harlow, Kenneth W.; Nygaard, Per; Hove-Jensen, Bjarne

    1995-01-01

    The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell...

  16. Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role

    Czech Academy of Sciences Publication Activity Database

    Dobrová, Zuzana; Sardanelli, A. M.; Speranza, F.; Scacco, S.; Signorile, A.; Lorusso, V.; Papa, S.

    2001-01-01

    Roč. 40, - (2001), s. 13941-13947 ISSN 0006-2960 Institutional research plan: CEZ:AV0Z5020903 Keywords : cAMP * cyclic adenosine monophosphate Subject RIV: CE - Biochemistry Impact factor: 4.114, year: 2001

  17. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    Science.gov (United States)

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt

    2016-06-01

    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

    Science.gov (United States)

    Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

    2016-11-01

    2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. The guanosine nucleotide (p)ppGpp initiates development and A-factor production in myxococcus xanthus.

    Science.gov (United States)

    Harris, B Z; Kaiser, D; Singer, M

    1998-04-01

    Guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp], synthesized in response to amino acid limitation, induce early gene expression leading to multicellular fruiting body formation in Myxococcus xanthus. A mutant (DK527) that fails to accumulate (p)ppGpp in response to starvation was found to be blocked in development prior to aggregation. By use of a series of developmentally regulated Tn5lac transcriptional fusion reporters, the time of developmental arrest in DK527 was narrowed to within the few hours of development, the period of starvation recognition. The mutant is also defective in the production of A-factor, an early extracellular cell-density signal. The relA gene from Escherichia coli, which encodes a ribosome-dependent (p)ppGpp synthetase, rescues this mutant. We also demonstrate that inactivation of the M. xanthus relA homolog blocks development and the accumulation of (p)ppGpp. Moreover, the wild-type allele of Myxococcus relA rescues DK527. These observations support a model in which accumulation of (p)ppGpp, in response to starvation, initiates the program of fruiting body development, including the production of A-factor.

  20. Mechanism of formation of (deoxy)guanosine adducts derived from peroxidase-catalyzed oxidation of the carcinogenic nonaminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I)

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Cvačka, Josef; Semanská, M.; Martínek, V.; Frei, E.; Stiborová, M.

    2009-01-01

    Roč. 22, č. 11 (2009), s. 1765-1773 ISSN 0893-228X R&D Projects: GA AV ČR KJB400550903 Grant - others:GA ČR(CZ) GA303/09/0472; GA ČR(CZ) GA203/09/0812 Institutional research plan: CEZ:AV0Z40550506 Keywords : Sudan I * peroxidase * NMR spectroscopy * (deoxy)guanosin adducts Subject RIV: CE - Biochemistry Impact factor: 3.740, year: 2009

  1. Structural Characterization of the Molecular Events during a Slow Substrate-Product Transition in Orotidine 5'-Monophosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Fujihashi, Masahiro; Wei, Lianhu; Kotra, Lakshmi P; Pai, Emil F; (TGRI); (Toronto); (Kyoto)

    2009-04-06

    Crystal structures of substrate-product complexes of Methanobacterium thermoautotrophicum orotidine 5'-monophosphate decarboxylase, obtained at various steps in its catalysis of the unusual transformation of 6-cyano-uridine 5'-monophosphate (UMP) into barbituric acid ribosyl monophosphate, show that the cyano substituent of the substrate, when bound to the active site, is first bent significantly from the plane of the pyrimidine ring and then replaced by an oxygen atom. Although the K72A and D70A/K72A mutants are either catalytically impaired or even completely inactive, they still display bending of the C6 substituent. Interestingly, high-resolution structures of the D70A and D75N mutants revealed a covalent bond between C6 of UMP and the Lys72 side chain after the -CN moiety's release. The same covalent bond was observed when the native enzyme was incubated with 6-azido-UMP and 6-iodo-UMP; in contrast, the K72A mutant transformed 6-iodo-UMP to barbituric acid ribosyl 5'-monophosphate. These results demonstrate that, given a suitable environment, native orotidine 5'-monophosphate decarboxylase and several of its mutants are not restricted to the physiologically relevant decarboxylation; they are able to catalyze even nucleophilic substitution reactions but consistently maintain distortion on the C6 substituent as an important feature of catalysis.

  2. Investigations of structural, dielectric and optical properties on silicon ion irradiated glycine monophosphate single crystals

    Energy Technology Data Exchange (ETDEWEB)

    Kanagasekaran, T. [Department of Physics, Anna University, Chennai 600 025 (India); Department of Physics and Astrophysics, University of Delhi, New Delhi 110 007 (India); Mythili, P. [Department of Physics, Anna University, Chennai 600 025 (India); Bhagavannarayana, G. [Materials Characterization Division, National Physical Laboratory, New Delhi 110012 (India); Kanjilal, D. [Inter University Accelerator Centre, New Delhi 110 067 (India); Gopalakrishnan, R. [Department of Physics, Anna University, Chennai 600 025 (India)], E-mail: krgkrishnan@annauniv.edu

    2009-08-01

    The 50 MeV silicon ion irradiation induced modifications on structural, optical and dielectric properties of solution grown glycine monophosphate (GMP) crystals were studied. The high-resolution X-ray diffraction study shows the unaltered value of integrated intensity on irradiation. The dielectric constant as a function of frequency and temperature was studied. UV-visible studies reveal the decrease in bandgap values on irradiation and presence of F-centers. The fluorescence spectrum shows the existence of some energy levels, which remains unaffected after irradiation. The scanning electron micrographs reveal the defects formed on irradiation.

  3. Structure of Radicals from X-irradiated Guanine Derivatives: An Experimental and Computational Study of Sodium Guanosine Dihydrate Single Crystals

    Science.gov (United States)

    Jayatilaka, Nayana; Nelson, William H.

    2008-01-01

    In sodium guanosine dihydrate single crystals, the guanine moiety is deprotonated at N1 due to growth from high-pH (>12) solutions. EPR and ENDOR study of crystals x-irradiated at 10 K detected evidence for three radical forms. Radical R1,characterized by two proton and two nitrogen hyperfine interactions, was identified as the product of net hydrogenation at N7 of the N1-deprotonated guanine unit. R1 exhibited an unusually distorted structure leading to net positive isotropic components of the hydrogen couplings. Radical R2, characterized by one proton and one nitrogen hyperfine coupling was identified as the primary electron loss product. This product is equivalent to that of deprotonation at N1 by the guanine cation and represents the first ENDOR characterization of that product. Radical R3, characterized by a single hydrogen hyperfine coupling, was identified as the product of net dehydrogenation at C1 of the ribose moiety. The identification of radicals R1-R3 was supported by DFT calculations on several possible structures using the B3LYP/6-311G(2df,p)//6-31G(d,p) approach. Radical R4, detected after warming the crystals to room temperature, was identified as the well-known product of net hydrogenation of C8 of the (N1-deprotonated) guanine component. Radical R1, evidently formed by protonation of the primary electron addition product, was present as roughly 60% of the total radicals detected at 10 K. Radical R2 was present as roughly 27% of the total yield, and the concentration of R3 contributed the remaining 13%. R3 is evidently the product of oneelectron oxidation followed by deprotonation; thus, the balance of oxidation and reduction products is approximately equal within experimental uncertainty. PMID:17249824

  4. Method of preparing thymidine-5'-monophosphate specifically or nonspecifically labelled with 14C or with 3H

    International Nuclear Information System (INIS)

    Nejedly, Z.; Filip, J.; Ekl, J.; Kolina, J.; Votruba, I.; Skoda, J.

    1977-01-01

    The invention claims a method for labelled thymidine-5'-monophosphate preparation by cultivating a special thymine-dependent Escherichia coli SPT - strain in the optimum synthetic culture medium containing 0.8 to 1.2 g/ml of labelled thymine. Practically the whole amount of labelled thymine is utilized for cellular deoxyribonucleic acid synthesis. The radioactive biomass obtained is processed using such chemical and enzymatic decomposition procedures as to allow separating the labelled thymidine-5'-monophosphate as the only thymine reaction product. Experiments conducted showed that the radiochemical purity of the thymidine-5'-monophosphate obtained was better than 98%. The absence of other nonactive substances was confirmed by spectrophotometric analysis. The overall product activity was 92.3% of the activity of thymine-2- 14 C introduced in the reaction. (Ha)

  5. Direct Determination of Six Cytokinin Nucleotide Monophosphates in Coconut Flesh by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry.

    Science.gov (United States)

    Cao, Zhao-Yun; Ma, You-Ning; Sun, Li-Hua; Mou, Ren-Xiang; Zhu, Zhi-Wei; Chen, Ming-Xue

    2017-11-15

    Coconut contains many uncharacterized cytokinins that have important physiological effects in plants and humans. In this work, a method based on liquid chromatography-tandem mass spectrometry was developed for identification and quantification of six cytokinin nucleotide monophosphates in coconut flesh. Excellent separation was achieved using a low-coverage C18 bonded-phase column with an acidic mobile phase, which greatly improved the retention of target compounds. To enable high-throughput analysis, a single-step solid-phase extraction using mixed-mode anion-exchange cartridges was employed for sample preparation. This proved to be an effective method to minimize matrix effects and ensure high selectivity. The limits of detection varied from 0.06 to 0.3 ng/mL, and the limits of quantification ranged from 0.2 to 1.0 ng/mL. The linearity was statistically verified over 2 orders of magnitude, giving a coefficient of determination (R 2 ) greater than 0.9981. The mean recoveries were from 81 to 108%; the intraday precision (n = 6) was less than 11%; and the interday precision (n = 11) was within 14%. The developed method was applied to the determination of cytokinin nucleotide monophosphates in coconut flesh samples, and four of them were successfully identified and quantified. The results showed that trans-zeatin riboside-5'-monophosphate was the dominant cytokinin, with a concentration of 2.7-34.2 ng/g, followed by N 6 -isopentenyladenosine-5'-monophosphate (≤12.9 ng/g), while the concentrations of cis-zeatin riboside-5'-monophosphate and dihydrozeatin riboside-5'-monophosphate were less than 2.2 and 4.9 ng/g, respectively.

  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. Kinetics of hydrogen-deuterium exchange in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) determined by laser-Raman spectroscopy.

    Science.gov (United States)

    Thomas, G J; Livramento, J

    1975-11-18

    Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) (poly(rA)) were determined as a function of temperature in the range 20-90 degrees C by means of laser-Raman spectroscopy. For 5'-rAMP, the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature, i.e., kpsi = Ae-Ea/RT, with A = 2.3 X 10(14) hr-1 and Ea = 24.2 +/- 0.6 kcal/mol. For cAMP, above 50 degrees C, kpsi is nearly identical in magnitude and temperature dependence to that of 5'-rAMP. However, below 50 degrees C, isotope exchange in cAMP is much more rapid than in 5'-rAMP, characterized by a lower activation energy (17.7 kcal/mol) and frequency factor (9.6 X 10(9) hr-1). Exchange in poly(rA) is considerably slower than in 5'-rAMP at all temperatures, but like cAMP the in k vs. 1/T plot may be divided into high temperature and low temperature domains, each characterized by different Arrhenius parameters. Above 60 degrees C, poly(rA) gives Ea = 22.0 kcal/mol and A = 3.2 X 10(12) hr-1, while below 60 degrees C, Ea = 27.7 kcal/mol and A = 1.8 X 10(16) hr-1. Thus, increasing the temperature above 60 degrees C does not diminish the retardation of exchange in poly(rA) vis a vis 5'-rAMP. These results indicate that the distribution of electrons in the adenine ring of cAMP is altered by lowering the temperature below 50 degrees C, although no similar perturbation occurs for 5'-rAMP. Retardation of exchange in poly(rA) is most probably due to base stacking at lower temperatures and to steric hindrance from the ribopolymer backbone at higher temperatures. We also report the spectral effects of deuterium exchange on the vibrational Raman frequencies of 5'-rAMP, cAMP, and poly(rA) and suggest a number of new assignments for the 5' and cyclic ribosyl phosphate groups.

  8. Therapeutic effects of the joint administration of magnesium aspartate and adenosine monophosphate in gamma-irradiated mice

    International Nuclear Information System (INIS)

    Pospisil, M.; Netikova, J.; Pipalova, I.; Kozubik, A.

    1990-01-01

    The joint administration of magnesium aspartate and adenosine monophosphate, injected on days 1 to 4 post radiation, has been found to exert stimulatory effects on the recovery of hemopoietic functions in sublethally gamma-irradiated mice. These therapeutical effects were enhanced in animals protected by peroral administration of cystamine. The treatment scheme used did not modify survival of lethally irradiated mice. The therapeutic effects of magnesium aspartate and adenosine monophosphate in sublethally irradiated mice are explained by the stimulatory action of these drugs on the cell adenylate cyclase system, which influences the erythropoietic functions. (author)

  9. 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

    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,

  10. The solvent effect on the enthalpy of hydrolysis of cyclic adenosine 3',5'-monophosphate : a quantum chemical study

    NARCIS (Netherlands)

    Scheffers - Sap, Miek; Buck, H.M.

    1978-01-01

    The solvent effect on the enthalpy of hydrolysis has been studied by the Extended-Hückel method for the hydrolysis reactions of cyclic adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) and related cyclic phosphate diesters. The results show that the difference in enthalpy of hydrolysis between cyclic

  11. Inhibitors of inosine monophosphate dehydrogenase: SARs about the N-[3-Methoxy-4-(5-oxazolyl)phenyl moiety.

    Science.gov (United States)

    Iwanowicz, Edwin J; Watterson, Scott H; Guo, Junqing; Pitts, William J; Murali Dhar, T G; Shen, Zhongqi; Chen, Ping; Gu, Henry H; Fleener, Catherine A; Rouleau, Katherine A; Cheney, Daniel L; Townsend, Robert M; Hollenbaugh, Diane L

    2003-06-16

    The first reported structure-activity relationships (SARs) about the N-[3-methoxy-4-(5-oxazolyl)phenyl moiety for a series of recently disclosed inosine monophosphate dehydrogenase (IMPDH) inhibitors are described. The syntheses and in vitro inhibitory values for IMPDH II, and T-cell proliferation (for select analogues) are given.

  12. Hydrogels Based on Ag+ -Modulated Assembly of 5'-Adenosine Monophosphate for Enriching Biomolecules.

    Science.gov (United States)

    Hu, Yuanyuan; Xie, Dong; Wu, Yang; Lin, Nangui; Song, Aixin; Hao, Jingcheng

    2017-11-07

    Supramolecular hydrogels obtained by combining 5'-adenosine monophosphate (AMP) with Ag + were fabricated in this work. Their gelation capability was enhanced by increasing the concentration of Ag + or decreasing the pH. The gels are very sensitive to light, which endows them with potential applications as visible-light photosensitive materials. Coordination between the nucleobase of AMP and Ag + , as well as π-π stacking of nucleobases, are considered to be the main driving forces for self-assembly. The hydrogels successfully achieved the encapsulation and enrichment of biomolecules. Hydrogen bonding between the amino group of guest molecules and silver nanoparticles along the nanofibers drives the enrichment and is considered to be a crucial interaction. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

    Science.gov (United States)

    Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

    2008-01-01

    Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

  14. DNA-specific labelling by deoxyribonucleoside 5'-monophosphates in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Brendel, M.; Faeth, W.W.; Toper, R.

    1975-01-01

    Growth of 5'-dTMP low-requiring strains is inhibited by exogenous 5'-dGMP and 5'-GMP at concentrations higher than 5 x 10 -4 M. Synthesis of nucleic acids ceases and cells remain fixed in their respective place in the cell cycle. At concentrations lower than 10 -5 M deoxyribonucleoside 5'-monophosphates may be employed for radioactive labelling, the label being preferentially used for DNA synthesis. Affinity to DNA of the 5'-dNMPs is in the order of 5'-dAMPS > 5'-dGMP > 5'-dCMP > 5'-dUMP. DNA-specific label is achieved with 5'-dAMP when the medium is supplemented with adenine and deoxyadenosine. (orig.) [de

  15. Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate

    International Nuclear Information System (INIS)

    Barbieri, J.T.; Collins, C.M.; Collier, R.J.

    1986-01-01

    Diphtheria toxin (DT) that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap. BS-C-1 cells were incubated with or without 125 I-DT or CRM 197. They were then incubated with [ 32 P]ApUp, and assayed

  16. Enhancement of radioprotective effectiveness of adenosine monophosphate by magnesium aspartate in mice

    International Nuclear Information System (INIS)

    Pospisil, M.; Netikova, J.; Kozubik, A.; Chertkov, K.S.; Ministry of Health, Moscow

    1988-01-01

    The enhancing effect of magnesium aspartate on the radioprotective effectiveness of adenosine monophosphate (AMP) administered to whole-body gamma-irradiated mice was studied. Male (CBA x C57BL/10)F 1 hybrid mice of a mean body weight of 32 g were used. 5 mg AMP per mouse was injected i.p. 15 min before and 15 min after irradiation; magnesium aspartate (13.3 mg per mouse) was administered s.c. 35 min before irradiation. The benefical effect of the drug combination used was manifested when investigating hematological indices at the recovery phase of sublethally irradiated animals, as well as when observing the survival of lethally irradiated mice. The synergistic radioprotective effects of AMP and magnesium aspartate are explained by the stimulatory action of both these compounds on the cell adenylate cyclase system. (author)

  17. Effect of sildenafil on acrolein-induced airway inflammation and mucus production in rats.

    Science.gov (United States)

    Wang, T; Liu, Y; Chen, L; Wang, X; Hu, X-R; Feng, Y-L; Liu, D-S; Xu, D; Duan, Y-P; Lin, J; Ou, X-M; Wen, F-Q

    2009-05-01

    Airway inflammation with mucus overproduction is a distinguishing pathophysiological feature of many chronic respiratory diseases. Phosphodiesterase (PDE) inhibitors have shown anti-inflammatory properties. In the present study, the effect of sildenafil, a potent inhibitor of PDE5 that selectively degrades cyclic guanosine 3',5'-monophosphate (cGMP), on acrolein-induced inflammation and mucus production in rat airways was examined. Rats were exposed to acrolein for 14 and 28 days. Sildenafil or distilled saline was administered intragastrically prior to acrolein exposure. Bronchoalveolar lavage fluid (BALF) was acquired for cell count and the detection of pro-inflammatory cytokine levels. Lung tissue was examined for cGMP content, nitric oxide (NO)-metabolite levels, histopathological lesion scores, goblet cell metaplasia and mucin production. The results suggested that sildenafil pretreatment reversed the significant decline of cGMP content in rat lungs induced by acrolein exposure, and suppressed the increase of lung NO metabolites, the BALF leukocyte influx and pro-inflammatory cytokine release. Moreover, sildenafil pretreatment reduced acrolein-induced Muc5ac mucin synthesis at both mRNA and protein levels, and attenuated airway inflammation, as well as epithelial hyperplasia and metaplasia. In conclusion, sildenafil could attenuate airway inflammation and mucus production in the rat model, possibly through the nitric oxide/cyclic guanosine 3',5'-monophosphate pathway, and, thus, might have a therapeutic potential for chronic airway diseases.

  18. Involvement of NO/cGMP pathway in the antidepressant-like effect of gabapentin in mouse forced swimming test.

    Science.gov (United States)

    Ostadhadi, Sattar; Kordjazy, Nastaran; Haj-Mirzaian, Arya; Ameli, Sanaz; Akhlaghipour, Golnoosh; Dehpour, AhmadReza

    2016-04-01

    Based on clinical studies regarding the beneficial effect of gabapentin in depression, we aimed to evaluate the antidepressant-like properties of gabapentin in mice and also the participation of nitric oxide (NO)/cyclic guanosine monophosphate pathway in this effect. The following drugs were used in this study: gabapentin; N(G)-nitro-L-arginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor; 7-nitroindazole, a specific neuronal NOS inhibitor; aminoguanidine, a specific inducible NOS inhibitor; L-arginine, a NO precursor; and sildenafil, a phosphodiestrase inhibitor. Finally, we studied the behavioral effects through the forced swimming test (FST) and the changes of the hippocampus NO level through nitrite assay. The immobility time was significantly reduced after gabapentin administration. Co-administration of non-effective doses of gabapentin and L-NAME or 7-nitroindazole (7-NI) resulted in antidepressant-like effect in FST, while aminoguanidine did not affect the immobility time of gabapentin-treated mice. Furthermore, the antidepressant-like property of gabapentin was prevented by L-arginine or sildenafil. Also, the hippocampal nitrite level was significantly lower in gabapentin-treated mice relative to saline-injected mice, and co-administration of 7-NI with sub-effective gabapentin caused a significant decrease in hippocampal nitrite levels. Our results indicate that the antidepressant-like effect of gabapentin in the mice FST model is mediated at least in part through nitric oxide/cyclic guanosine monophosphate (cGMP) pathway.

  19. Chemical synthesis of guanosine diphosphate mannuronic acid (GDP-ManA) and its C-4-O-methyl and C-4-deoxy congeners.

    Science.gov (United States)

    Zhang, Qingju; Howell, P Lynne; Overkleeft, Herman S; Filippov, Dmitri V; van der Marel, Gijsbert A; Codée, Jeroen D C

    2017-10-10

    Described is the first synthesis of guanosine diphosphate mannuronic acid (GDP-ManA), the sugar donor used by algae and bacteria for the production of alginate, an anionic polysaccharide composed of β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). Understanding the biosynthesis of these polyanionic polysaccharides on the molecular level, opens up avenues to use and modulate the biosynthesis machinery for biotechnological and therapeutic applications. The synthesis reported here delivers multi-milligram amounts of the GDP-ManA donor that can be used to study the polymerase (Alg8 in Pseudomonas aeruginosa) that generates the poly-ManA chain. Also reported is the assembly of two close analogues of GDP-ManA: the first bears a C-4-O-methyl group, while the second has been deoxygenated at this position. Both molecules may be used as "chain stoppers" in future enzymatic ManA polymerisation reactions. The crucial pyrophosphate linkage of the GDP-mannuronic acids has been constructed by the phosphorylation of the appropriate ManA-1-phosphates with a guanosine phosphoramidite. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Low Intensity Extracorporeal Shock Wave Therapy Improves Erectile Function in a Model of Type II Diabetes Independently of NO/cGMP Pathway.

    Science.gov (United States)

    Assaly-Kaddoum, Rana; Giuliano, François; Laurin, Miguel; Gorny, Diane; Kergoat, Micheline; Bernabé, Jacques; Vardi, Yoram; Alexandre, Laurent; Behr-Roussel, Delphine

    2016-09-01

    Erectile dysfunction is highly prevalent in type II diabetes mellitus. Low intensity extracorporeal shock wave therapy improves erectile function in patients with erectile dysfunction of vasculogenic origin, including diabetes. However, its mode of action remains unknown. We investigated the effects of low intensity extracorporeal shock wave therapy compared to or combined with sildenafil on erectile dysfunction in a type II diabetes mellitus model. Our purpose was to test our hypothesis of a mode of action targeting the cavernous nitric oxide/cyclic guanosine monophosphate pathway. GK rats, a validated model of type II diabetes mellitus, and age matched Wistar rats were treated with low intensity extracorporeal shock wave therapy twice weekly for 3 weeks. Treatment was repeated after a 3-week no-treatment interval. The penis was stretched and dipped in a specifically designed water-filled cage. Shock waves were delivered by a calibrated probe yielding a controlled energy flux density (0.09 mJ/mm(2)). The probe was attached to an electrohydraulic unit with a focused shock wave source, allowing for accurate extrapolation to humans. Following a 4-week washout period erectile function was assessed as well as endothelium dependent and independent, and nitrergic relaxations of the corpus cavernosum of GK rats. Low intensity extracorporeal shock wave therapy significantly improved erectile function in GK rats to the same extent as sildenafil. Treatment effects were potentiated when combined with sildenafil. Shock wave effects were not associated with improved cavernous endothelium dependent or independent, or nitrergic reactivity. Low intensity extracorporeal shock wave therapy improved erectile function in GK rats. Unexpectedly, this was not mediated by a nitric oxide/cyclic guanosine monophosphate dependent mechanism. Sildenafil increased shock wave efficacy. This preclinical paradigm to deliver low intensity extracorporeal shock wave therapy to the rat penis should

  1. Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

    Science.gov (United States)

    Yang, Ke-Ke; Sui, Yi; Zhou, Hui-Rong; Zhao, Hai-Lu

    2017-05-01

    Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and

  2. (5'-32P)-8-azidoguanosine-3',5'-monophosphate. I. Synthesis and properties. II. Interaction with E. coli proteins

    International Nuclear Information System (INIS)

    Owens, J.R.

    1983-01-01

    Under certain conditions of nutritional deprivation, microorganisms produce the magic spot nucleotides guanosine-3'-diphosphate-5'-triphosphate(pppGpp) and the tetraphosphate ppGpp. The latter is known to be a pleiotypic effector, i.e. it inhibits (and sometimes stimulates) many biological processes including transcription, translation, and metabolic pathways. It is unknown whether pppGpp, ppGp, pGpp, and pGp, other members of this family of guanosine-3',5'-phosphates, also have regulatory properties. To begin to investigate this question, a radioactive photoaffinity analog of pGp was prepared: (5' 32 P)pN 3 Gp. The interaction of this photoprobe with E. coli sonicates and a purified protein (RNA polymerase) was examined. At physiological salt concentrations two proteins (RNA polymerase) was examined. At physiological salt concentrations two proteins of 86,000 and 65,000 daltons (p86 and p65) were primarily photolabeled. Competition studies with guanosine and adenosine nucleotides indicated (5 32 P)pN 3 Gp was labeling a ppGpp binding site on p86, and a pGp (or GMP) site on p65. ATP phosphorylation of p86 increased photoincorporation, while it decreased labeling of p65. The data also provide evidence of a different type of regulatory mechanism, i.e. phosphorylation modulates binding of an allosteric effector (ppGpp) to a protein(enzyme). Both ATP and GTP were found to phosphorylate the same proteins, although GTP was the preferred substrate in some cases

  3. Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

    International Nuclear Information System (INIS)

    Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

    2006-01-01

    Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V M = 2.3 Å 3 Da −1 )

  4. Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

    Science.gov (United States)

    Mitamura, Toshiaki; Yamamura, Yoshimi; Kurosaki, Fumiya

    2011-01-01

    Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.

  5. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    Science.gov (United States)

    Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2008-11-01

    A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

  6. Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

    Science.gov (United States)

    Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K N; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad; Cortopassi, Wilian A; Chandler, Shane A; Rasmussen, Tim; Benesch, Justin L P; Paton, Robert S; Claridge, Timothy D W; Miller, Samantha; Booth, Ian R; Naismith, James H; Conway, Stuart J

    2017-08-15

    Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

  7. 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.

  8. Identification of the orotidine-5'-monophosphate decarboxylase gene of the oleaginous yeast Rhodosporidium toruloides.

    Science.gov (United States)

    Yang, Fan; Zhang, Sufang; Tang, Wei; Zhao, Zongbao K

    2008-09-01

    Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer of great industrial potential, yet there is no effective genetic tool for rationally engineering this microorganism. To develop a marker recycling system, the orotidine-5'-monophosphate (OMP) decarboxylase gene of R. toruloides (RtURA3) was isolated using methods of degenerate polymerase chain reaction (PCR) together with rapid amplification of cDNA ends. The results showed that RtURA3 contains four extrons and three introns, and that the encoded polypeptide holds a sequence of 279 amino acid residues with significant homology to those of OMP decarboxylases from other yeasts. A shuttle vector pYES2/CT-RtURA3 was constructed via site-specific insertion of RtURA3 into the commercial vector pYES2/CT. Transformation of the shuttle vector into Saccharomyces cerevisiae BY4741, a URA3-deficient yeast strain, ensured the viability of the strain on synthetic dextrose agar plate without uracil, suggesting that the isolated RtURA3 was functionally equivalent to the URA3 gene from S. cerevisiae.

  9. Inosine-5'-monophosphate is a candidate agent to resolve rigor mortis of skeletal muscle.

    Science.gov (United States)

    Matsuishi, Masanori; Tsuji, Mariko; Yamaguchi, Megumi; Kitamura, Natsumi; Tanaka, Sachi; Nakamura, Yukinobu; Okitani, Akihiro

    2016-11-01

    The object of the present study was to reveal the action of inosine-5'-monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19-0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0-3.3 m mol/L adenosine-5'-diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP-induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments. © 2016 Japanese Society of Animal Science.

  10. A Mixed-Valent Molybdenum Monophosphate with a Layer Structure: KMo 3P 2O 14

    Science.gov (United States)

    Guesdon, A.; Borel, M. M.; Leclaire, A.; Grandin, A.; Raveau, B.

    1994-03-01

    A new mixed-valent molybdenum monophosphate with a layer structure KMo 3P 2O 14 has been isolated. It crystallizes in the space group P2 1/ m with a = 8.599(2) Å, b = 6.392(2) Å, c = 10.602(1) Å, and β = 111.65(2)°. The layers [Mo 3P 2O 14] ∞ are parallel to (100) and consist of [MoPO 8] ∞ chains running along limitb→ , in which one MoO 6 octahedron alternates with one PO 4 tetrahedron. In fact, four [MoPO 8] ∞ chains share the corners of their polyhedra and the edges of their octahedra, forming [Mo 4P 4O 24] ∞ columns which are linked through MoO 5 bipyramids along limitc→. The K + ions interleaved between these layers are surrounded by eight oxygens, forming bicapped trigonal prisms KO 8. Besides the unusual trigonal bipyramids MoO 5, this structure is also characterized by a tendency to the localization of the electrons, since one octahedral site is occupied by Mo(V), whereas the other octahedral site and the trigonal bipyramid are occupied by Mo(VI). The similarity of this structure with pure octahedral layer structures suggests the possibility of generating various derivatives, and of ion exchange properties.

  11. Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia

    Science.gov (United States)

    Santoso; Thornburg

    1998-02-01

    To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

  12. Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1

    Science.gov (United States)

    Santoso, Djoko; Thornburg, Robert

    1998-01-01

    To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells. PMID:9490773

  13. Hit discovery of Mycobacterium tuberculosis inosine 5'-monophosphate dehydrogenase, GuaB2, inhibitors.

    Science.gov (United States)

    Sahu, Niteshkumar U; Singh, Vinayak; Ferraris, Davide M; Rizzi, Menico; Kharkar, Prashant S

    2018-04-18

    Tuberculosis remains a global concern. There is an urgent need of newer antitubercular drugs due to the development of resistant forms of Mycobacterium tuberculosis (Mtb). Inosine 5'-monophosphate dehydrogenase (IMPDH), guaB2, of Mtb, required for guanine nucleotide biosynthesis, is an attractive target for drug development. In this study, we screened a focused library of 73 drug-like molecules with desirable calculated/predicted physicochemical properties, for growth inhibitory activity against drug-sensitive MtbH37Rv. The eight hits and mycophenolic acid, a prototype IMPDH inhibitor, were further evaluated for activity on purified Mtb-GuaB2 enzyme, target selectivity using a conditional knockdown mutant of guaB2 in Mtb, followed by cross-resistance to IMPDH inhibitor-resistant SRMV2.6 strain of Mtb, and activity on human IMPDH2 isoform. One of the hits, 13, a 5-amidophthalide derivative, has shown growth inhibitory potential and target specificity against the Mtb-GuaB2 enzyme. The hit, 13, is a promising molecule with potential for further development as an antitubercular agent. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Crystal Structure of the Thermus thermophilus 16 S rRNA Methyltransferase RsmC in Complex with Cofactor and Substrate Guanosine

    Energy Technology Data Exchange (ETDEWEB)

    Demirci, H.; Gregory, S; Dahlberg, A; Jogl, G

    2008-01-01

    Post-transcriptional modification is a ubiquitous feature of ribosomal RNA in all kingdoms of life. Modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. Here we describe high resolution crystal structures for the N{sup 2}-guanine methyltransferase RsmC that modifies residue G1207 in 16 S rRNA near the decoding site of the 30 S ribosomal subunit. RsmC is a class I S-adenosyl-l-methionine-dependent methyltransferase composed of two methyltransferase domains. However, only one S-adenosyl-l-methionine molecule and one substrate molecule, guanosine, bind in the ternary complex. The N-terminal domain does not bind any cofactor. Two structures with bound S-adenosyl-l-methionine and S-adenosyl-l-homocysteine confirm that the cofactor binding mode is highly similar to other class I methyltransferases. Secondary structure elements of the N-terminal domain contribute to cofactor-binding interactions and restrict access to the cofactor-binding site. The orientation of guanosine in the active site reveals that G1207 has to disengage from its Watson-Crick base pairing interaction with C1051 in the 16 S rRNA and flip out into the active site prior to its modification. Inspection of the 30 S crystal structure indicates that access to G1207 by RsmC is incompatible with the native subunit structure, consistent with previous suggestions that this enzyme recognizes a subunit assembly intermediate.

  15. Clustering of nucleosides in the presence of alkali metals: Biologically relevant quartets of guanosine, deoxyguanosine and uridine observed by ESI-MS/MS.

    Science.gov (United States)

    Aggerholm, Tenna; Nanita, Sergio C; Koch, Kim J; Cooks, R Graham

    2003-01-01

    Electrospray ionization (ESI) mass spectra of nucleosides, recorded in the presence of alkali metals, display alkali metal ion-bound quartets and other clusters that may have implications for understanding non-covalent interactions in DNA and RNA. The tetramers of guanosine and deoxyguanosine and also their metaclusters (clusters of clusters), cationized by alkali metals, were observed as unusually abundant magic number clusters. The observation of these species in the gas phase parallels previous condensed-phase studies, which show that guanine derivatives can form quartets and metaclusters of quartets in solution in the presence of metal cations. This parallel behavior and also internal evidence suggest that bonding in the guanosine tetramers involves the bases rather than the sugar units. The nucleobases thymine and uracil are known to form magic number pentameric adducts with K+, Cs+ and NH4+ in the gas phase. In sharp contrast, we now show that the nucleosides uridine and deoxythymidine do not form the pentameric clusters characteristic of the corresponding bases. More subtle effects of the sugars are evident in the fact that adenosine and cytidine form numerous higher order clusters with alkali metals, whereas deoxyadenosine and deoxycytidine show no clustering. It is suggested that hydrogen bonding between the bases in the tetramers of dG and rG are the dominant interactions in the clusters, hence changing the ribose group to deoxyribose (and vice versa) generally has little effect. However, the additional hydroxyl group of RNA nucleosides enhances the non-selective formation of higher-order aggregates for adenosine and cytidine and results in the lack of highly stable magic number clusters. Some clusters are the result of aggregation in the course of ionization (ESI) whereas others appear to be intrinsic to the solution being examined. Copyright 2003 John Wiley & Sons, Ltd.

  16. 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.

  17. Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

    Science.gov (United States)

    McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

    2017-07-07

    NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Novel adenosine 3',5'-cyclic monophosphate dependent protein kinases in a marine diatom

    International Nuclear Information System (INIS)

    Lin, P.P.C.; Volcani, B.E.

    1989-01-01

    Two novel adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg 2+ and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser( 32 P)-Ser-Asn-Ala-Arg and have an apparent M r of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M r of about 78,000 is photolabeled with 8-azido[ 32 P]cAMP and is also phosphorylated with [γ- 32 P]ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids

  19. Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

    Science.gov (United States)

    Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

    2008-02-01

    Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

  20. Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

    Science.gov (United States)

    Goldman, Lawrence M; Amyes, Tina L; Goryanova, Bogdana; Gerlt, John A; Richard, John P

    2014-07-16

    The mechanism for activation of orotidine 5'-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters k(cat) and K(m) for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-D-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (k(cat))(obs) for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (K(m))(obs).

  1. Further studies on the effect of adenosine cyclic monophosphate derivatives on cell proliferation in the jejunal crypts of rat.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1982-01-01

    1. Cell proliferation in the jejunal crypt epithelium of rat was measured using a stathmokinetic technique. 2. Sodium butyrate was found to promote jejunal crypt cell proliferation. 3. N6, O2'-Dibutyryl cyclic adenosine monophosphate (cAMP), N6-monobutyryl-cAMP and N6-monobutyryl-8-bromo-cAMP were found to inhibit cell proliferation when compared to sodium butyrate treated tissues. 4. 8-Chlorophenylthio-cAMP was found to inhibit cell division when compared to untreated animals. 5. O2'-Monobutyryl cAMP and 8-bromo-cAMP were not found to inhibit cell proliferation.

  2. An interplay among FIS, H-NS and guanosine tetraphosphate modulates transcription of the Escherichia coli cspA gene under physiological growth conditions

    Directory of Open Access Journals (Sweden)

    Anna eBrandi

    2016-05-01

    Full Text Available CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((pppGpp and in vitro transcription assays, we show that the cspA promoter is sensitive to (pppGpp inhibition. The (pppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription.

  3. Cloning and characterization of a gene encoding Rac/Rop-like monomeric guanosine 5'-triphosphate-binding protein from Scoparia dulcis.

    Science.gov (United States)

    Mitamura, Toshiaki; Shite, Masato; Yamamura, Yoshimi; Kurosaki, Fumiya

    2009-06-01

    A cDNA clone, designated Sd-racrop (969 bp), was isolated from seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 197 amino acid residues with high homology to Rac/Rop small guanosine 5'-triphosphate-binding proteins from various plant sources. In Southern hybridization analysis, the restriction digests prepared from genomic DNA of S. dulcis showed a main signal together with a few weakly hybridized bands. The transcriptional level of Sd-racrop showed a transient decrease by exposure of the leaf tissues of S. dulcis to the ethylene-generating reagent 2-chloroethylphosphonic acid. However, an appreciable increase in gene expression was reproducibly observed upon treatment of the plant with methyl jasmonate. These results suggest that the Sd-racrop product plays roles in ethylene- and methyl jasmonate-induced responses of S. dulcis accompanying the change in the transcriptional level, however, the cellular events mediated by this protein toward these external stimuli would be regulated by various mechanisms.

  4. The effect of adenosine 5'-monophosphate (AMP) on tenderness, microstructure and chemical-physical index of duck breast meat.

    Science.gov (United States)

    Wang, Daoying; Deng, Shaoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong

    2016-03-30

    Adenosine 5'-monophosphate (AMP) is often used in meat and poultry soups as a flavor enhancer (flavor modifier), or as food additives for specific nutritional purposes. Our previous research as well as evidence from others showed that actomyosin could be dissociated into myosin and actin by AMP in extracted muscle solution. However, there is no report available on the application of AMP to dissociate actomyosin and to improve meat tenderness. The objectives of this study were to evaluate the effect of AMP on duck meat tenderness and other quality traits and to explore the mechanism of the action of AMP on meat tenderness. Duck breast muscle was treated with 0, 10, 20, 30, 40 mmol L(-1) AMP at 5 °C for 10 h and examined for shear force, microstructure, actomyosin dissociation, myofibril fragmentation index (MFI), pH, water content, cooking loss, CIE* color (L*, a*, b*), inosine monophosphate (IMP) and free amino acid (FAA) contents. Results showed that shear force, cooking loss, L* and b* of the muscles significantly decreased after AMP treatment (P 0.05), and muscle shrinkage in transverse and longitudinal directions were restrained after AMP treatment. The results suggest that AMP could notably improve meat tenderness, and this effect was probably mainly through increasing muscle pH, promoting actomyosin dissociation and disrupting the Z-line; meanwhile, the conversion of AMP to IMP may contribute to the flavor of meat. © 2015 Society of Chemical Industry.

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

    International Nuclear Information System (INIS)

    Han, Byung Woo; Bingman, Craig A.; Mahnke, Donna K.; Sabina, Richard L.; Phillips, George N. Jr

    2005-01-01

    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 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

  6. 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.

  7. Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

    Science.gov (United States)

    Chao, Julie; Weathersbee, Carolyn J.

    1974-01-01

    Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

  8. Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine

    Science.gov (United States)

    Profy, A. T.; Usher, D. A.

    1984-01-01

    The aminoacylation of diinosine monophosphate was studied experimentally. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40 percent enantiomeric excess of the isomer was incorporated at the 2' site and the positions of equilibrium for the reversible 2'-3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups was found to decrease on the order 2'(3') less than internal 2' and less than 5', and the extent of the reaction was affected by the concentration of the imidazole buffer. Reaction of IpI with imidazolide of unprotected DL-alanine, by contrast, led to an excess of the D isomer at the internal 2' site. Finally, reaction with the N-carboxy anhydride of DL-alanine occurred without stereoselection. These results are found to be relevant to the study of the evolution of optical chemical activity and the origin of genetically directed protein synthesis.

  9. Radiorestoring activity of few nucleotides on normal tissues of Jerusalem Artichoke after an irradiation with γ rays of 60Co

    International Nuclear Information System (INIS)

    Jonard, Robert; Bayonove, Jacqueline; Riedel, Michel.

    1978-01-01

    The nucleotides tested: adenosine triphosphate (ATP) and cyclic adenosine 3',5'-monophosphate (3',5'-cAMP), guanosine triphosphate (GTP) and cyclic guanosine 3',5'-monophosphate (3',5'-cGMP), are able to restore proliferation to irradiated (γ irradiation, 3,000 rad) Jesusalem Artichoke tissue. The 3',5'-cGMP shows the greater radiorestoring activity [fr

  10. The effects of L-arginine, D-arginine, L-name and methylene blue on channa striatus-induced peripheral antinociception in mice.

    Science.gov (United States)

    Zakaria, Zainul Amiruddin; Sulaiman, Mohd Rosian; Somchit, Muhammad Nazrul; Jais, Abdul Manan Mat; Ali, Daud Israf

    2005-08-03

    To determine the involvement of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway in aqueous supernatant of haruan (Channa striatus) fillet (ASH) antinociception using the acetic acid-induced abdominal constriction test. The ASH was prepared by soaking fresh haruan fillet in chloroform:methanol (CM) (2/1 (v/v)) for 72 h followed by evaporation of the upper layer supernatant to remove any solvent residues. The supernatant was then subjected to a freeze-drying process (48 h) followed by doses preparation. Subcutaneous (SC) administration of ASH alone (0.170, 0.426 and 1.704 mg/kg) exhibited a dose-dependent antinociception. On the other hand, 20 mg/kg (SC) of L-arginine and MB exhibited a significant nociception and antinociception, while D-arginine and L-NAME did not produce any effect at all. Pre-treatment with L-arginine was found to significantly reverse the three respective doses of ASH antinociception; pre-treatment with D-arginine did not produce any significant change in the ASH activity; pre-treatment with L-NAME only significantly increased the 0.170 and 0.426 mg/kg ASH antinociception; and pre-treatment with MB significantly enhanced the respective doses of ASH antinociception, respectively. Furthermore, co-treatment with L-NAME significantly enhanced the L-arginine reversal effect on 0.426 mg/kg ASH antinociception. In addition, MB significantly reversed the L-arginine nociception on 0.426 mg/kg ASH. These finding suggest ASH antinociception involves the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. The presence of NO was found to reverse ASH antinociceptive activity while blocking of cGMP system enhanced it.

  11. Orotidine-5'-monophosphate decarboxylase catalysis: Kinetic isotope effects and the state of hybridization of a bound transition-state analogue

    Energy Technology Data Exchange (ETDEWEB)

    Acheson, S.A.; Bell, J.B.; Jones, M.E.; Wolfenden, R. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))

    1990-04-03

    The enzymatic decarboxylation of orotidine 5'-monophosphate may proceed by an addition-elimination mechanism involving a covalently bound intermediate or by elimination of CO2 to generate a nitrogen ylide. In an attempt to distinguish between these two alternatives, 1-(phosphoribosyl)barbituric acid was synthesized with 13C at the 5-position. Interaction of this potential transition-state analogue inhibitor with yeast orotidine-5'-monophosphate decarboxylase resulted in a small (0.6 ppm) downfield displacement of the C-5 resonance, indicating no rehybridization of the kind that might have been expected to accompany 5,6-addition of an enzyme nucleophile. When the substrate orotidine 5'-monophosphate was synthesized with deuterium at C-5, no significant change in kcat (H/D = 0.99 +/- 0.06) or kcat/KM (H/D = 1.00 +/- 0.06) was found to result, suggesting that C-5 does not undergo significant changes in geometry before or during the step that determines the rate of the catalytic process. These results are consistent with a nitrogen ylide mechanism and offer no support for the intervention of covalently bound intermediates in the catalytic process.

  12. A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates

    International Nuclear Information System (INIS)

    Randerath, Kurt; Randerath, Erika; Danna, T.F.; Van Golen, K.L.; Putman, K.L.

    1989-01-01

    A new sensitive 32 P-postlabelling assay for DNA adducts has been developed. When DNA containing bulky adducts, X 1 , X 2 , .....X n , is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pX i pN, because inter-nucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, X i pN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'- 32 P-labeled dinucleotides,[ 32 P]pX i pN, by T4 polynucleotide kinase-catalyzed [ 32 P]posphate transfer from [γ- 32 P]ATP. Upon mapping on polyethyleneimine-cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, [ 32 P]pX i and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new 32 P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'- 32 P-labeled 3',5'-bisphosphates, [ 32 P]pX i p. (author)

  13. Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5′-monophosphate

    International Nuclear Information System (INIS)

    Shi, Fan; Gong, Shixing; Xu, Li; Zhu, Huanhuan; Sun, Zhenfan; Sun, Wei

    2013-01-01

    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. - Highlights: • A graphene, ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite were prepared. • Composite-modified carbon molecular wire electrode was fabricated and characterized. • A sensitive electrochemical method for the detection of adenosine-5′-monophosphate was established

  14. Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5′-monophosphate

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Fan [Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158 (China); Gong, Shixing; Xu, Li; Zhu, Huanhuan [College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China); Sun, Zhenfan [Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158 (China); Sun, Wei, E-mail: swyy26@hotmail.com [Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158 (China)

    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. - Highlights: • A graphene, ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite were prepared. • Composite-modified carbon molecular wire electrode was fabricated and characterized. • A sensitive electrochemical method for the detection of adenosine-5′-monophosphate was established.

  15. Bronchodilator responses after methacholine and adenosine 5'-monophosphate (AMP) challenges in children with asthma: their relationships with eosinophil markers.

    Science.gov (United States)

    Yoo, Young; Seo, Sung Chul; Kim, Young Il; Chung, Bo Hyun; Song, Dae Jin; Choung, Ji Tae

    2012-09-01

    Bronchodilator responsiveness (BDR) and eosinophilic inflammation are characteristic features of asthma. Objective. The aim of this study was to compare the relationships of BDR after methacholine challenge or adenosine 5'-monophosphate (AMP) challenge to blood eosinophil markers in children with asthma. Methacholine and AMP challenges were performed on 69 children with mild intermittent to moderate persistent asthma. BDR was calculated as the change in forced expiratory volume in 1 second, expressed as percentage change of the value immediately after the each challenge and the value after inhalation of salbutamol. Serum total IgE levels, blood eosinophil counts, and serum eosinophil cationic protein (ECP) levels were determined for each subject. A positive relationship between serum total IgE levels and BDR was found only after the AMP challenge (R(2) = 0.345, p = .001) rather than after the methacholine challenge (R(2) = 0.007, p = .495). Peripheral blood eosinophil counts correlated more significantly with BDR after AMP challenge (R(2) = 0.212, p = .001) than BDR after methacholine challenge (R(2) = 0.002, p = .724). Both BDR after methacholine challenge (R(2) = 0.063, p = .038) and BDR after AMP challenge (R(2) = 0.192, p = .001) were significantly correlated with serum ECP levels. BDR after AMP challenge may be more closely related to eosinophilic inflammation, compared with that after methacholine challenge.

  16. Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

    International Nuclear Information System (INIS)

    Smiley, J.A.; Bell, J.B.; Jones, M.E.; Paneth, P.; O'Leary, M.H.

    1991-01-01

    Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13 C kinetic isotope effect of 1.0247 ± 0.0008 at 25 C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 ± 0.0006 measured at pH 4.0, 25 C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel, which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. These data fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegal

  17. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  18. 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

    2008-01-01

    The propensity of four representative conformations of 2'-deoxyadenosine-5'-monophosphate (5'-dAMPH) to bind an excess electron has been studied at the B3LYP /6-31++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&barbelow;south-syn anion is 1.19eV, while its vertical detachment energy is 1.89eV. 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.

  19. Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

    Science.gov (United States)

    Chen, Kan; Cao, Wanlu; Li, Juan; Sprengers, Dave; Hernanda, Pratika Y; Kong, Xiangdong; van der Laan, Luc JW; Man, Kwan; Kwekkeboom, Jaap; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-01-01

    As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA. PMID:26467706

  20. Experimental measurement and modelling of solubility of inosine-5′-monophosphate disodium in pure and mixed solvents

    International Nuclear Information System (INIS)

    Zou, Fengxia; Zhuang, Wei; Wu, Jinglan; Zhou, Jingwei; Liu, Qiyan; Chen, Yong; Xie, Jingjing; Zhu, Chenjie; Guo, Ting; Ying, Hanjie

    2014-01-01

    Graphical abstract: - Highlights: • Solubility of 5′-IMPNa 2 in various solvents was studied for the first time. • The solubility could be ranked as follows: water > methanol > ethanol > acetone. • Modified Apelblat equation gave the best correlating results. • Mixing Gibbs free energies, enthalpies, and entropies were predicted. • Solubility data and equations can optimise the crystallization conditions. - Abstract: The solubility of biological chemicals in solvents provide important fundamental data and is generally considered as an essential factor in the design of crystallization processes. The equilibrium solubility data of inosine-5′-monophosphate disodium (5′-IMPNa 2 ) in water, methanol, ethanol, acetone, as well as in the solvent mixtures (methanol + water, ethanol + water, acetone + water), were measured by an isothermal method at temperatures ranging from (293.15 to 313.15) K. The measured data in pure and mixed solvents were then modelled using the modified Apelblat equation, van’t Hoff equation, λh equation, ideal model and the Wilson model. The modified Apelblat equation showed the best modelling results, and it was therefore used to predict the mixing Gibbs free energies, enthalpies, and entropies of 5′-IMPNa 2 in pure and binary solvents. The positive values of the calculated partial molar Gibbs free energies indicated the variations in the solubility trends of 5′-IMPNa 2 . Water and ethanol (in the binary mixture with water) were found to be the most effective solvent and anti-solvent, respectively

  1. The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

    Science.gov (United States)

    Hsieh, Wei-Yu; Liao, Jo-Chien; Wang, Hsin-Tzu; Hung, Tzu-Huan; Tseng, Ching-Chih; Chung, Tsui-Yun; Hsieh, Ming-Hsiun

    2017-07-01

    Thiamin diphosphate (TPP, vitamin B 1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  2. Adenosine monophosphate is not superior to histamine for bronchial provocation test for assessment of asthma control and symptoms.

    Science.gov (United States)

    Wu, Fan; Guan, Wei-Jie; Gao, Yi; An, Jia-Ying; Xie, Yan-Qing; Liu, Wen-Ting; Yu, Xin-Xin; Zheng, Jin-Ping

    2017-07-01

    Adenosine monophosphate (AMP) may reflect airway inflammation and hyperresponsiveness, but relationship between AMP and histamine (His, a conventional stimulus) bronchial provocation test (BPT) in asthma is not fully elucidated. To compare both BPTs and determine their utility in reflecting changes of asthmatic symptoms. BPTs were performed in a cross-over fashion, at 2-4 day intervals. Cumulative doses eliciting 20% FEV 1 fall (PD 20 FEV 1 ), diagnostic performance and adverse events (AEs) were compared. Patients with PD 20 FEV 1 lower than geometric mean were defined as responders, otherwise poor responders. Patients with uncontrolled and partly controlled asthma, who maintained their original inhaled corticosteroids therapy, underwent reassessment of airway responsiveness and asthmatic symptoms 3 and 6 months after. Nineteen uncontrolled, 22 partly controlled and 19 controlled asthmatic patients and 24 healthy subjects were recruited. Lower PD 20 FEV 1 geometric means were associated with poorer asthma control in His-BPT (0.424 μmol vs 1.684 μmol vs 3.757 μmol), but not AMP-BPT (11.810 μmol vs 7.781 μmol vs 10.220 μmol). Both BPTs yielded similar overall diagnostic performance in asthma (area under curve: 0.842 in AMP-BPT vs 0.850 in His-BPT). AEs, including wheezing and tachypnea, were similar and mild. Ten patients with uncontrolled and 10 partly controlled asthma were followed-up. At months 3 and 6, we documented an increase in PD 20 FEV 1 -AMP and PD 20 FEV 1 -His, which did not correlate with reduction asthmatic symptom scores. This overall applied in responders and poor responders of AMP-BPT and His-BPT. Despite higher screening capacity of well-controlled asthma, AMP-BPT confers similar diagnostic performance and safety with His-BPT. AMP-BPT might not preferentially reflect changes asthmatic symptoms. © 2015 John Wiley & Sons Ltd.

  3. 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. © 2011 John Wiley & Sons A/S.

  4. Relationships of methacholine and adenosine 5'-monophosphate (AMP) responsiveness to the postbronchodilator FEV₁/FVC ratio in children with asthma.

    Science.gov (United States)

    Suh, Dong In; Choi, Sun Hee; Lee, Ju Kyung; Kim, Jin-Tack; Koh, Young Yull

    2011-05-01

    Airway remodeling has been assumed to cause bronchial hyperresponsiveness (BHR). A low postbronchodilator FEV₁/FVC ratio has been suggested to be a functional surrogate marker of airway remodeling in asthma. BHR is commonly assessed by bronchial challenges using direct or indirect stimuli. The aim of this study was to compare BHR to methacholine and adenosine 5'-monophosphate (AMP) with regard to their relationship with a marker of airway remodeling in children with asthma. Methacholine and AMP challenge tests were performed in 129 children with asthma, aged 12 years, and a provocative concentration causing a 20% fall in FEV₁ (PC₂₀) was calculated for each challenge. All subjects also underwent pre- and postbronchodilator spirometry. A postbronchodilator FEV₁/FVC ratio below the lower limits of normal was used as a marker of airway remodeling. A low postbronchodilator FEV₁/FVC ratio was found in 17 subjects (13.2%). These subjects had a significantly lower methacholine PC₂₀ (geometric mean: 0.63 mg/mL, range of 1 SD: 0.17-2.29) than those (n = 112) with a normal postbronchodilator FEV₁/FVC ratio (2.42 mg/mL, 0.57-10.32, p = .000), whereas AMP PC₂₀ was similar between the two groups (22.1 mg/mL, 3.9-125.9 vs. 27.7 mg/mL, 4.2-183.5, p = .231). In the whole group of subjects, methacholine PC₂₀, but not AMP PC₂₀, correlated significantly with the postbronchodilator FEV₁/FVC ratio (r = 0.340, p = .000, and r = 0.056, p = .526, respectively). Our results provide evidence, though indirect, that BHR to methacholine is related to airway remodeling in children with asthma and suggest that BHR to methacholine may be a better marker of airway remodeling than BHR to AMP.

  5. Effects of exogenous inosine monophosphate on growth performance, flavor compounds, enzyme activity, and gene expression of muscle tissues in chicken.

    Science.gov (United States)

    Yan, Junshu; Liu, Peifeng; Xu, Liangmei; Huan, Hailin; Zhou, Weiren; Xu, Xiaoming; Shi, Zhendan

    2018-04-01

    The goal of this experiment was to examine effects of diets supplemented with exogenous inosine monophosphate (IMP) on the growth performance, flavor compounds, enzyme activity and gene expression of chicken. A total of 1,500 healthy, 1-day-old male 3-yellow chickens were used for a 52-d experimental period. Individuals were randomly divided into 5 groups (group I, II, III, IV, V) with 6 replicates per group, and fed a basal diet supplemented with 0.0, 0.05, 0.1, 0.2, and 0.3% IMP, respectively. There was no significant response to the increasing dietary IMP level in average daily feed intake (ADFI), average daily gain (ADG), and feed:gain ratio (F/G) (P ≥ 0.05). IMP content of the breast and thigh muscle showed an exponential and linear response to the increasing dietary IMP level (P exogenous IMP was fed. There were significant effects of IMP level in diet on free amino acids (FAA) (exponential, linear and quadratic effect, P exogenous IMP was fed. Dietary IMP supplementation had a quadratic effect on 5΄-NT and the alkaline phosphatase (ALP) enzyme activity in the breast muscle (P exogenous IMP group had the highest (AMPD1) gene expression of the breast muscle and ATIC gene expression of the thigh muscle. These results indicate that dietary IMP did not affect the growth performance of chicken, the diet with 0.2 to 0.3% exogenous IMP is optimal to improve the meat flavor quality in chicken.

  6. Ginsenoside Compound K suppresses the hepatic gluconeogenesis via activating adenosine-5'monophosphate kinase: A study in vitro and in vivo.

    Science.gov (United States)

    Wei, Shengnan; Li, Wei; Yu, Yang; Yao, Fan; A, Lixiang; Lan, Xiaoxin; Guan, Fengying; Zhang, Ming; Chen, Li

    2015-10-15

    Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenoside. We have reported that CK presented anti-diabetic effect via diminishing the expressions of hepatic gluconeogenesis key enzyme. Here, we further explore the possible mechanism of CK on suppression hepatic gluconeogenesis via activation of adenosine-5'monophosphate kinase (AMPK) on type 2 diabetes mice in vivo and in HepG2 cells. Type 2 diabetes mice model was developed by high fat diet combined with STZ injection. 30mg/kg/d CK was orally administrated for 4weeks, the fasting blood glucose level and 2h OGTT were conducted, and the protein expression of AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) were examined. The mechanism of Compound K on hepatic gluconeogenesis was further explored in HepG2 hepatocytes. Glucose production, the protein expression of AMPK, PEPCK, G6pase and PGC-1α, hepatic nuclear factor 4α (HNF-4α) and forkhead transcription factor O1 (FOXO1) were determined after Compound K treatment at the presence of AMPK inhibitor Compound C. We observed that CK inhibited the expression of PEPCK and G6Pase in the liver and in HepG2 hepatocytes. Meanwhile, CK treatment remarkably increased the activation of AMPK, while decreasing the expressions of PGC-1α, HNF-4α and FOXO1. However, AMPK inhibitor Compound C could reverse these effects of CK on gluconeogenesis in part. The results indicated that the effect of CK on suppression hepatic gluconeogenesis might be via the activation the AMPK activity. Copyright © 2015. Published by Elsevier Inc.

  7. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    International Nuclear Information System (INIS)

    Delamere, N.A.; Socci, R.R.; King, K.L.

    1990-01-01

    The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin

  8. Erythrocyte metabolism in hyperthyroidism: a microcalorimetric study on changes in the Embden-Meyerhof and the hexose monophosphate pathways.

    Science.gov (United States)

    Monti, M; Hedner, P; Ikomi-Kumm, J; Valdemarsson, S

    1987-05-01

    Erythrocyte metabolism was studied in vitro by microcalorimetry in 10 hyperthyroid subjects before and after treatment. By inhibiting the enzyme enolase in the Embden-Meyerhof pathway with sodium fluoride (NaF) we have recorded the anaerobic and aerobic contributions in erythrocyte thermogenesis. The decrease in heat production rate in samples with NaF corresponds to the anaerobic contribution, whereas the values from samples with NaF reflect aerobic processes. Before treatment, total heat production rate was 120 +/- 2 mW/l erythrocytes which was higher than the post-treatment value of 99 +/- 2 (P less than 0.001) as well as the value for 14 euthyroid subjects, 108 +/- 2 mW/l (P less than 0.001). The NaF inhibitable rate was 73 +/- 2 before and 63 +/- 1 mW/l after therapy (P less than 0.01). These values correspond to 61 +/- 1 and 64 +/- 1% (n.s.) of the total heat production rate, and were similar to that of 61 +/- 2% for the controls. Heat production rates in the presence of NaF were 47 +/- 1 before and 36 +/- 1 mW/l after therapy (P less than 0.001), representing 39 +/- 1 and 36 +/- 1% of total values, respectively. The present results show that overall metabolism is increased in erythrocytes from hyperthyroid subjects before treatment and returns to normal after normalization of the thyroid function. Moreover, by using microcalorimetry we found that the metabolic activity along the Embden-Meyerhof anaerobic pathway as well as along the hexose monophosphate aerobic pathway in erythrocytes is stimulated by thyroid hormones.

  9. Sugar-modified G-quadruplexes: effects of LNA-, 2′F-RNA– and 2′F-ANA-guanosine chemistries on G-quadruplex structure and stability

    Science.gov (United States)

    Li, Zhe; Lech, Christopher Jacques; Phan, Anh Tuân

    2014-01-01

    G-quadruplex-forming oligonucleotides containing modified nucleotide chemistries have demonstrated promising pharmaceutical potential. In this work, we systematically investigate the effects of sugar-modified guanosines on the structure and stability of a (4+0) parallel and a (3+1) hybrid G-quadruplex using over 60 modified sequences containing a single-position substitution of 2′-O-4′-C-methylene-guanosine (LNAG), 2′-deoxy-2′-fluoro-riboguanosine (FG) or 2′-deoxy-2′-fluoro-arabinoguanosine (FANAG). Our results are summarized in two parts: (I) Generally, LNAG substitutions into ‘anti’ position guanines within a guanine-tetrad lead to a more stable G-quadruplex, while substitutions into ‘syn’ positions disrupt the native G-quadruplex conformation. However, some interesting exceptions to this trend are observed. We discover that a LNAG modification upstream of a short propeller loop hinders G-quadruplex formation. (II) A single substitution of either FG or FANAG into a ‘syn’ position is powerful enough to perturb the (3+1) G-quadruplex. Substitution of either FG or FANAG into any ‘anti’ position is well tolerated in the two G-quadruplex scaffolds. FANAG substitutions to ‘anti’ positions are better tolerated than their FG counterparts. In both scaffolds, FANAG substitutions to the central tetrad layer are observed to be the most stabilizing. The observations reported herein on the effects of LNAG, FG and FANAG modifications on G-quadruplex structure and stability will enable the future design of pharmaceutically relevant oligonucleotides. PMID:24371274

  10. Characterization of radiation-induced products of thymidine 3'-monophosphate and thymidylyl (3'→5') thymidine by high-performance liquid chromatography and laser-desorption fourier-transform mass spectrometry

    International Nuclear Information System (INIS)

    Yoshida, H.; Hettich, R.L.

    1994-01-01

    High-performance liquid chromatography (HPLC) and laser-desorption Fourier-transform mass spectrometry (LD FTMS) have been applied for direct measurements of radiation-induced products of nucleic acid constituents containing thymidine. Laser desorption FTMS could be used for the direct detection (neither hydrolyzed nor derivatized) of X-ray-induced decomposition products of aqueous thymidine monophosphate. After these initial experiments, a variety of hydrogenated and hydroxylated thymine standards were acquired and examined by FTMS to assist in the identification of unknown radiation-induced decomposition products of thymine-containing nucleotides and dinucleotides. To extend these studies to dinucleotides, the radiation-induced products generated by the gamma radiolysis of thymidylyl (3'→5') thymidine (TpT) were isolated by reverse-phase HPLC and identified by LD FTMS. Thymine and thymidine 3'-monophosphate were observed as the major products in this case. Several of the minor products of the HPLC profile were pooled in a single fraction and characterized simultaneously by LD FTMS. The resulting mass spectra indicated the presence of hydroxy-5,6-dihydothymidine monophosphate, 5,6-dihydrothymidine monophosphate and thymidine monophosphate, thymine glycol, hydroxy-5,6-dihydrothymine, 5-hydroxy-methyl-uracil and 5,6-dihydrothymine. The combination of HPLC purification and LD FTMS structural characterization provides a useful tool for the direct measurement of radiation-induced products of nucleotides and dinucleotides. 28 refs., 6 figs., 2 tabs

  11. Behavior of the monophosphate tungsten bronzes (PO2)4(WO3)2m (m = 7 and 8) in the course of electrochemical lithium insertion

    International Nuclear Information System (INIS)

    Martinez-de la Cruz, A.; Longoria Rodriguez, F.E.; Gonzalez, Lucy T.; Torres-Martinez, Leticia M.

    2007-01-01

    The electrochemical lithium insertion process has been studied in the family of monophosphate tungsten bronzes (PO 2 ) 4 (WO 3 ) 2m , where m = 7 and 8. 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 analyzed. The nature of the bronzes Li x (PO 2 ) 4 (WO 3 ) 2m formed was determined by in situ X-ray diffraction experiments. These results have allowed establishing a correlation with the reversible/irreversible processes detected during the electrochemical lithium insertion

  12. Effects of adenosine monophosphate on induction of therapeutic hypothermia and neuronal damage after cardiopulmonary resuscitation in rats.

    Science.gov (United States)

    Knapp, Jürgen; Schneider, Andreas; Nees, Corinna; Bruckner, Thomas; Böttiger, Bernd W; Popp, Erik

    2014-09-01

    Animal studies and pathophysiological considerations suggest that therapeutic hypothermia after cardiopulmonary resuscitation is the more effective the earlier it is induced. Therefore this study is sought to examine whether pharmacological facilitated hypothermia by administration of 5'-adenosine monophosphate (AMP) is neuroprotective in a rat model of cardiac arrest (CA) and resuscitation. Sixty-one rats were subjected to CA. After 6 min of ventricular fibrillation advanced cardiac life support was started. After successful return of spontaneous circulation (ROSC, n=40), animals were randomized either to placebo group (n=14) or AMP group (800 mg/kg body weight, n=14). Animals were kept at an ambient temperature of 18°C for 12 h after ROSC and core body temperature was measured using a telemetry temperature probe. Neuronal damage was analyzed by counting Nissl-positive (i.e. viable) neurons and TUNEL-positive (i.e. apoptotic) cells in coronal brain sections 7 days after ROSC. Functional status evaluated on days 1, 3 and 7 after ROSC by a tape removal test. Time until core body temperature dropped to <34.0°C was 31 min [28; 45] in AMP-treated animals and 125 min [90; 180] in the control group (p=0.003). Survival until 7 days after ROSC was comparable in both groups. Also number of Nissl-positive cells (AMP: 1 [1; 7] vs. placebo: 2 [1; 3] per 100 pixel; p=0.66) and TUNEL-positive cells (AMP: 56 [44; 72] vs. placebo: 53 [41; 67] per 100 pixel; p=0.70) did not differ. Neither did AMP affect functional neurological outcome up to 7 days after ROSC. Mean arterial pressure 20 min after ROSC was 49 [45; 55] mmHg in the AMP group in comparison to 91 [83; 95] mmHg in the control group (p<0.001). Although application of AMP reduced the time to reach a core body temperature of <34°C neither survival was improved nor neuronal damage attenuated. Reason for this is probably induction of marked hypotension as an adverse reaction to AMP treatment. Copyright © 2014 Elsevier

  13. 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.

  14. OXIDACIÓN DE p -NITROFENOL USANDO TiO 2 -ADENOSINA MONOFOSFATO I OXIDATION OF p -NITROPHENOL USING TiO 2 -ADENOSIN MONOPHOSPHATE

    Directory of Open Access Journals (Sweden)

    Carlos F. Rivas

    2018-04-01

    Full Text Available The surface of TiO2 was modified with the nucleotides adenosine 3’-monophosphate (AMP’3 and Adenosine 5’-monophosphate (AMP’5. The adsorption of nucleotides was adjusted to Langmuir ́s adsorption model, determining that the optimal condition for TiO 2 modification was at neutral pH. UV-Visible Diffuse Reflectance and IR Attenuated Total Reflectance spectra show that the chemisorption of nucleotides take placed on TiO 2 anatase. The new catalysts (TiO 2 -nucleotide improved the photodegradation of p -nitrophenol in a wide range of pH as compared with the titanium dioxide precursor. Most photoactivity was generated by using the new photocatalytic in the degradation of p -nitrophenol at pH = 6, obtaining high values for the pseudo first order kinetic constant (0.0254 min -1 and 0.0244 min -1 for TiO 2 -AMP’3 and TiO 2 -AMP’5, respectively. For all pH, the trend obtained for the photodegradation was: TiO 2 -AMP ́3 @ TiO 2 -AMP’5 > TiO 2 . Langmuir-Hinshelwood kinetics shows that the contribution of the surface reac tion rate governs the oxidation of the contaminant.

  15. Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling.

    Science.gov (United States)

    Okajima, F; Tomura, H; Sho, K; Kimura, T; Sato, K; Im, D S; Akbar, M; Kondo, Y

    1997-01-01

    Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.

  16. Bronchial responsiveness to adenosine-5 '-monophosphate and methacholine as predictors for nasal symptoms due to newly introduced allergens. A follow-up study among laboratory animal workers and bakery apprentices

    NARCIS (Netherlands)

    de Meer, G; Postma, DS; Heederik, D

    Background In asthma patients, bronchial hyper-responsiveness (BHR) to adenosine-5'-monophosphate (AMP) reflects bronchial inflammation more closely than BHR to methacholine. In this follow-up study we studied bronchial responsiveness to both stimuli as predictors of new-onset airway symptoms.

  17. SIMULTANEOUS ANALYSIS OF AZIDOTHYMIDINE AND ITS MONOPHOSPHATE, DIPHOSPHATE AND TRIPHOSPHATE DERIVATIVES IN BIOLOGICAL-FLUIDS, TISSUE AND CULTURED-CELLS BY A RAPID HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD

    NARCIS (Netherlands)

    MOLEMA, G; JANSEN, RW; Visser, Jan; MEIJER, DKF

    1992-01-01

    A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro

  18. Measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine in cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Weimann, Allan; Simonsen, Anja Hviid; Poulsen, Henrik E

    2018-01-15

    Increased levels of nucleosides modified by oxidation in human cerebrospinal fluid (CSF) have several times been reported in Alzheimer patients and patients suffering from Parkinson's disease. The focus has especially been on nucleosides containing the 8-hydroxylation of guanine. Only few reports on quantification of the ribonucleoside 8-oxo-7,8-dihydro-guanosine (8oxoGuo) in CSF have been published, whereas more have been published on the quantification of the deoxy-ribonucleoside 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodGuo). The reports on the quantification of 8oxodGuo concentrations in CSF report absolute concentrations varying by a factor >10 5 in healthy humans. This could indicate that there is a serious specificity problem in some of the methods. In this paper an isotope-dilution UPLC-MS/MS method with high specificity and sensitivity for the quantification of 8oxoGuo and 8oxodGuo in CSF is presented. LLOQ for the two analytes is determined to 4pM and 2pM, respectively. The calibration curves has been tested to be linear in the range from 4 to 3,000pM for 8oxoGuo and between 2 and 3,000pM for 8oxodGuo. Using a weighting factor of 1/x the correlation coefficient "r" for both analytes is >0.999. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. 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.

  20. The influence of dibutyryl adenosine cyclic monophosphate on cell proliferation in the epithelium of the jejunal crypts, the colonic crypts and in colonic carcinomata of rat.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1980-01-01

    1. Cell proliferation in the jejunal crypts, the colonic crypts and in dimethylhydrazine (DMH)-induced adenocarcinomata of rat colon was measured using a stathmokinetic technique. 2. Dibutryl cyclic adneosine monophosphate (dibutyryl cAMP) was found to inhibit cell proliferation in colonic crypts and in colonic adenocarcinomata. 3. Dibutryl cAMP at very high doses was found to inhibit jejunal crypt cell proliferation but at lower doses was found to accelerate jejunal crypt cell proliferation. 4. Neither bilateral adrenalectomy nor chemical sympathectomy was found to abolish the ability of dibutryl cAMP to stimulate jejunal crypt cell proliferation. 5. The present results are difficult to interpret in terms of known hormonal influences on cell proliferation in the tissues examined and of established actions, of these hormones on cyclic nucleotide metabolism in other tissues.

  1. Characterization of free radicals in γ-irradiated polycrystalline uridine 5'-monophosphate: a study combining ESR, spin-trapping and HPLC

    International Nuclear Information System (INIS)

    Hiraoka, W.; Kuwabara, M.; Sato, F.

    1991-01-01

    Free radicals generated in γ-irradiated polycrystalline uridine 5'-monophosphate (5'-UMP) were studied by ESR, spin-trapping and high-performance liquid chromatography (HPLC). Although HPLC ultimately gave four spin-adducts, one component that was originally present disappeared during HPLC. Spin adducts due to two types of C6 radials were identified. One of these was thought to be formed by electron addition and subsequent protonation at the C6 position, and the other was presumed to be produced by electron addition and subsequent protonation at the O 4 position. The spin adducts derived from the C5 and C5' radicals were also identified. The spin adduct that disappeared during HPLC was thought to correspond to the C4'-centred radical. Computer simulation of ESR spectra was carried out to estimate the hyperfine splitting constants. (author)

  2. Studies of short-lived radicals in the. gamma. -irradiated aqueous solution of uridine-5'-monophosphate by the spin-trapping method and the liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Kominami, S [Hiroshima Univ. (Japan); Rokushika, S; Hatano, H

    1976-12-01

    An aerated aqueous solution of uridine-5'-monophosphate was ..gamma..-irradiated with 2-methyl-2-nitrosopropane as a spin-trapping reagent. Liquid chromatography was applied to separate the stable nitroxide radicals in the irradiated solution. The radicals were detected by U.V. and e.s.r. spectrometry. The e.s.r. detection showed four peaks in the chromatogram. The orcinol method for detection of the residual sugar moieties was applied before and after reduction of the base to determine the existence of the 5,6-double bond for the molecules in each fraction. From the combined results of the e.s.r. and orcinol methods, the short-lived radicals which were trapped by 2-methyl-2-nitrosopropane were identified as radicals of N-1 and C-6 positions of the base moiety and t-butyl radicals which was the radiolytic product of the trapping reagent.

  3. Modulation of growth performance, immunological responses and disease resistance of juvenile Nile tilapia (Oreochromis niloticus (Linnaeus, 1758 by supplementing dietary inosine monophosphate

    Directory of Open Access Journals (Sweden)

    Md. Abdul Kader

    2018-05-01

    Full Text Available This study was investigated to examine supplemental effects of dietary inosine monophosphate (IMP on growth performance, feed utilization, biochemical, hematological and immunological parameters of juvenile Nile tilapia Oreochromis niloticus. Disease resistance to experimental infection with Streptococcus agalactiae was also assessed. A semi-purified basal diet was supplemented with 0 (IMP0, Control, 1 (IMP1, 2 (IMP2, 4 (IMP4 and 8 (IMP8 g purified IMP kg−1 diet to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (0.59 g for 60 days. The results indicated that supplementation of IMP significantly (P  0.05. Among whole body proximate composition and somatic parameters, condition factor was significantly influenced by dietary supplementation of IMP. A wide variation in hematological parameters were observed and dietary supplementation increased the hematocrit content (P  0.05. Total serum protein (TSP, lysozyme activity (LA, superoxide dismutase activity (SOD and bactericidal activity (BA tended to increase with the supplementation of dietary IMP. TSP and SOD were significantly improved with ≥4 g kg−1 supplementation, while LA with 8 g kg−1 and BA with ≥1 g kg−1 supplementations. IMP supplemented groups showed higher (P > 0.05 cumulative survival compared to that of supplementation free control group. IMP supplemented diet groups also showed significantly higher BA in the post challenge test. Based on the overall performances, the results of the current study indicated that the inclusion of IMP in Nile tilapia diet can improve growth performance, feed utilization, haematological and immunological parameters; and disease resistance of juvenile Nile tilapia. Keywords: Nucleotides, Inosine monophosphate, Growth, Immunity, Disease resistance, Nile tilapia, Streptococcus agalactiae

  4. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    International Nuclear Information System (INIS)

    Lund, Kaleb C.; Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 μM), AZT monophosphate (150 μM), and 2',3'-dideoxycytidine (ddC; 1 μM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 μM) and ddC (1 μM). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-γ activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug

  5. Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

    Science.gov (United States)

    Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

    2015-06-01

    Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  6. Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

    Science.gov (United States)

    Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. Structure and dynamics of the ApA, ApC, CpA and CpC RNA dinucleoside monophosphates resolved with NMR scalar spin-spin couplings

    Czech Academy of Sciences Publication Activity Database

    Vokáčová, Zuzana; Schneider, Bohdan; Buděšínský, Miloš; Rosenberg, Ivan; Šponer, Jiří; Sychrovský, Vladimír

    2009-01-01

    Roč. 16, 1a (2009), b52-b52 ISSN 1211-5894. [Meeting of the Czechoslovak and Slovak Biologists /7./. 12.03.2009-14.03.2009, Nové Hrady] R&D Projects: GA AV ČR IAA400550701 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507 Keywords : NMR * dinucleoside monophosphate Subject RIV: CF - Physical ; Theoretical Chemistry

  8. Discovery of N-[2-[2-[[3-methoxy-4-(5-oxazolyl)phenyl]amino]-5-oxazolyl]phenyl]-N-methyl-4- morpholineacetamide as a novel and potent inhibitor of inosine monophosphate dehydrogenase with excellent in vivo activity.

    Science.gov (United States)

    Dhar, T G Murali; Shen, Zhongqi; Guo, Junqing; Liu, Chunjian; Watterson, Scott H; Gu, Henry H; Pitts, William J; Fleener, Catherine A; Rouleau, Katherine A; Sherbina, N Z; McIntyre, Kim W; Shuster, David J; Witmer, Mark R; Tredup, Jeffrey A; Chen, Bang-Chi; Zhao, Rulin; Bednarz, Mark S; Cheney, Daniel L; MacMaster, John F; Miller, Laura M; Berry, Karen K; Harper, Timothy W; Barrish, Joel C; Hollenbaugh, Diane L; Iwanowicz, Edwin J

    2002-05-23

    Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.

  9. 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

    2018-03-05

    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.

  10. 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. © 2015 Japanese Society of Animal Science.

  11. AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

    Science.gov (United States)

    Alves, Daiane S; Farr, Glen A; Seo-Mayer, Patricia; Caplan, Michael J

    2010-12-01

    The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

  12. Solubility of disodium cytidine 5′-monophosphate in different binary mixtures from 288.15 K to 313.15 K

    International Nuclear Information System (INIS)

    Yu, Jin; Ma, Tianle; Li, An; Chen, Xiaochun; Chen, Yong; Xie, Jingjing; Wu, Jinglan; Ying, Hanjie

    2013-01-01

    Highlights: • Solubility of 5′-CMPNa 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 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 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. Effects of octacosanol extracted from rice bran on blood hormone levels and gene expressions of glucose transporter protein-4 and adenosine monophosphate protein kinase in weaning piglets

    Directory of Open Access Journals (Sweden)

    Lei Long

    2015-12-01

    Full Text Available The object of this study was to explore the regulatory mechanism of octacosanol to the body of animals and the effects of octacosanol on blood hormone levels and gene expressions of glucose transporter protein (GLUT-4 and adenosine monophosphate protein kinase (AMPK in liver and muscle tissue of weaning piglets. A total of 105 crossbred piglets ([Yorkshire × Landrace] × Duroc with an initial BW of 5.70 ± 1.41 kg (21 d of age were used in a 6-wk trial to evaluate the effects of octacosanol and tiamulin supplementation on contents of triiodothyronine (T3, thyroxine (T4, growth hormone (GH, glucagon (GU and adrenaline (AD in blood and gene expressions of GLUT-4 and AMPK in liver and muscle. Piglets were randomly distributed into 3 dietary treatments on the basis of BW and sex. Each treatment had 7 replicate pens with 5 piglets per pen. Treatments were as followed: control group, tiamulin group and octacosanol group. The results showed that compared with control group and tiamulin group, octacosanol greatly promoted the secretion of T3, GH, GU and AD (P  0.05. Results of the present study has confirmed that octacosanol affects energy metabolism of body by regulating secretion of blood hormones and related gene expression in tissue of weaning piglets, which can reduce stress response and has an impact on performance.

  14. Adenosine monophosphate is elevated in the bronchoalveolar lavage fluid of mice with acute respiratory toxicity induced by nanoparticles with high surface hydrophobicity.

    Science.gov (United States)

    Dailey, Lea Ann; Hernández-Prieto, Raquel; Casas-Ferreira, Ana Maria; Jones, Marie-Christine; Riffo-Vasquez, Yanira; Rodríguez-Gonzalo, Encarnación; Spina, Domenico; Jones, Stuart A; Smith, Norman W; Forbes, Ben; Page, Clive; Legido-Quigley, Cristina

    2015-02-01

    Inhaled nanomaterials present a challenge to traditional methods and understanding of respiratory toxicology. In this study, a non-targeted metabolomics approach was used to investigate relationships between nanoparticle hydrophobicity, inflammatory outcomes and the metabolic fingerprint in bronchoalveolar fluid. Measures of acute lung toxicity were assessed following single-dose intratracheal administration of nanoparticles with varying surface hydrophobicity (i.e. pegylated lipid nanocapsules, polyvinyl acetate nanoparticles and polystyrene beads; listed in order of increasing hydrophobicity). Broncho-alveolar lavage (BAL) fluid was collected from mice exposed to nanoparticles at a surface area dose of 220 cm(2) and metabolite fingerprints were acquired via ultra pressure liquid chromatography-mass spectrometry-based metabolomics. Particles with high surface hydrophobicity were pro-inflammatory. Multivariate analysis of the resultant small molecule fingerprints revealed clear discrimination between the vehicle control and polystyrene beads (p < 0.05), as well as between nanoparticles of different surface hydrophobicity (p < 0.0001). Further investigation of the metabolic fingerprints revealed that adenosine monophosphate (AMP) concentration in BAL correlated with neutrophilia (p < 0.01), CXCL1 levels (p < 0.05) and nanoparticle surface hydrophobicity (p < 0.001). Our results suggest that extracellular AMP is an intermediary metabolite involved in adenine nucleotide-regulated neutrophilic inflammation as well as tissue damage, and could potentially be used to monitor nanoparticle-induced responses in the lung following pulmonary administration.

  15. Formation of Mixed-Ligand Complexes of Pd2+ with Nucleoside 5'-Monophosphates and Some Metal-Ion-Binding Nucleoside Surrogates

    Directory of Open Access Journals (Sweden)

    Oleg Golubev

    2014-10-01

    Full Text Available Formation of mixed-ligand Pd2+ complexes between canonical nucleoside 5'-monophosphates and five metal-ion-binding nucleoside analogs has been studied by 1H-NMR spectroscopy to test the ability of these nucleoside surrogates to discriminate between unmodified nucleobases by Pd2+-mediated base pairing. The nucleoside analogs studied included 2,6-bis(3,5-dimethylpyrazol-1-yl-, 2,6-bis(1-methylhydrazinyl- and 6-(3,5-dimethylpyrazol-1-yl-substituted 9-(β-d-ribofuranosylpurines 1–3, and 2,4-bis(3,5-dimethylpyrazol-1-yl- and 2,4-bis(1-methylhydrazinyl-substituted 5-(β-d-ribofuranosyl-pyrimidines 4–5. Among these, the purine derivatives 1-3 bound Pd2+ much more tightly than the pyrimidine derivatives 4, 5 despite apparently similar structures of the potential coordination sites. Compounds 1 and 2 formed markedly stable mixed-ligand Pd2+ complexes with UMP and GMP, UMP binding favored by 1 and GMP by 2. With 3, formation of mixed-ligand complexes was retarded by binding of two molecules of 3 to Pd2+.

  16. 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.

  17. 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.

  18. Pharmacokinetics of IDX184, a liver-targeted oral prodrug of 2'-methylguanosine-5'-monophosphate, in the monkey and formulation optimization for human exposure.

    Science.gov (United States)

    Pan-Zhou, Xin-Ru; Mayes, Benjamin A; Rashidzadeh, Hassan; Gasparac, Rahela; Smith, Steven; Bhadresa, Sanjeev; Gupta, Kusum; Cohen, Marita Larsson; Bu, Charlie; Good, Steven S; Moussa, Adel; Rush, Roger

    2016-10-01

    IDX184 is a phosphoramidate prodrug of 2'-methylguanosine-5'-monophosphate, developed to treat patients infected with hepatitis C virus. A mass balance study of radiolabeled IDX184 and pharmacokinetic studies of IDX184 in portal vein-cannulated monkeys revealed relatively low IDX184 absorption but higher exposure of IDX184 in the portal vein than in the systemic circulation, indicating >90 % of the absorbed dose was subject to hepatic extraction. Systemic exposures to the main metabolite, 2'-methylguanosine (2'-MeG), were used as a surrogate for liver levels of the pharmacologically active entity 2'-MeG triphosphate, and accordingly, systemic levels of 2'-MeG in the monkey were used to optimize formulations for further clinical development of IDX184. Capsule formulations of IDX184 delivered acceptable levels of 2'-MeG in humans; however, the encapsulation process introduced low levels of the genotoxic impurity ethylene sulfide (ES), which necessitated formulation optimization. Animal pharmacokinetic data guided the development of a tablet with trace levels of ES and pharmacokinetic performance equal to that of the clinical capsule in the monkey. Under fed conditions in humans, the new tablet formulation showed similar exposure to the capsule used in prior clinical trials.

  19. Free radical formation in deoxyguanosine-5'-monophosphate γ-irradiated in frozen solution. A computer-assisted analysis of temperature-dependent ESR spectra

    International Nuclear Information System (INIS)

    Gregoli, S.; Olast, M.; Bertinchamps, A.

    1977-01-01

    Deoxyguanosine-5'-monophosphate (dGMP) was γ-irradiated at 77 K in frozen aqueous solution and then annealed in a stepwise fashion up to the melting point. During this process, the primary radicals formed in DGMP at 77 K are progressively converted into secondary radical species. This is observed as changes in the spectrum intensity and conformation. Computer-assisted analysis of these temperature-dependent spectra permitted us to identify the transient radical species involved and to draw up single-radical concentration kinetics vs temperature. The radiation chemical behavior of dGMP was found to be quite similar to that of dAMP, investigated previously. In both these purine derivatives, radical anions are converted into radicals of H-addition to C-8, and radical cations are converted into radicals of OH-addition to the same position. In dGMP, however, the cationic channel is only induced under certain experimental conditions (alkaline pH, presence of electron scavengers). At neutral pH, G + radicals are quite stable and finally become deactivated without being converted into secondary GOH radicals. Specific deuterium substitution at carbon C-8, and irradiation in H 2 O or in D 2 O, confirmed that both H + and OH - attachments do occur at C-8, and that both the H + and OH - groups come from the aqueous medium

  20. Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

    Science.gov (United States)

    Zeden, Merve S; Schuster, Christopher F; Bowman, Lisa; Zhong, Qiyun; Williams, Huw D; Gründling, Angelika

    2018-03-02

    Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA , the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. 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. Copyright © 2011 Wiley Periodicals, Inc.

  2. Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major.

    Science.gov (United States)

    Hossain, Md Sakhawat; Koshio, Shunsuke; Ishikawa, Manabu; Yokoyama, Saichiro; Sony, Nadia Mahjabin

    2016-09-01

    Our study explored the dietary effects of adenosine monophosphate (AMP) to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream. A semi-purified basal diet supplemented with 0% (Control), 0.1% (AMP-0.1), 0.2% (AMP-0.2), 0.4% (AMP-0.4) and 0.8% (AMP-0.8) purified AMP to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4 g) for 56 days. The results indicated that dietary AMP supplements tended to improve growth performances. One of the best ones was found in diet group AMP-0.2, followed by diet groups AMP-0.1, AMP-0.4 and AMP-0.8. The Apparent digestibility coefficients (dry matter, protein and lipid) also improved by AMP supplementation and the significantly highest dry matter digestibility was observed in diet group AMP-0.2. Fish fed diet groups AMP-0.2 and AMP-0.4 had significantly higher peroxidase and bactericidal activities than fish fed the control diet. Nitro-blue-tetrazolium (NBT) activity was found to be significantly (P  0.05) by dietary supplementation. In contrast, catalase activity decreased with AMP supplementation. Moreover, the fish fed AMP supplemented diets had better improvement (P red sea bream. The regression analysis revealed that a dietary AMP supplementation between 0.2 and 0.4% supported weight gain and lysozyme activity as a marker of immune functions for red sea bream, which is also inline with the most of the growth and health performance parameters of fish under present experimental conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Overexpression of cyclic adenosine monophosphate effluent protein MRP4 induces an altered response to β-adrenergic stimulation in the senescent rat heart.

    Science.gov (United States)

    Carillion, Aude; Feldman, Sarah; Jiang, Cheng; Atassi, Fabrice; Na, Na; Mougenot, Nathalie; Besse, Sophie; Hulot, Jean-Sébastien; Riou, Bruno; Amour, Julien

    2015-02-01

    In the senescent heart, the positive inotropic response to β-adrenoceptor stimulation is reduced, partly by dysregulation of β1- and β3-adrenoceptors. The multidrug resistance protein 4 (MRP4) takes part in the control of intracellular cyclic adenosine monophosphate concentration by controlling its efflux but the role of MRP4 in the β-adrenergic dysfunction of the senescent heart remains unknown. The β-adrenergic responses to isoproterenol were investigated in vivo (stress echocardiography) and in vitro (isolated cardiomyocyte by Ionoptix with sarcomere shortening and calcium transient) in young (3 months old) and senescent (24 months old) rats pretreated or not with MK571, a specific MRP4 inhibitor. MRP4 was quantified in left ventricular homogenates by Western blotting. Data are mean ± SD expressed as percent of baseline value. The positive inotropic effect of isoproterenol was reduced in senescent rats in vivo (left ventricular shortening fraction 120 ± 16% vs. 158 ± 20%, P < 0.001, n = 16 rats) and in vitro (sarcomere shortening 129 ± 37% vs. 148 ± 35%, P = 0.004, n = 41 or 43 cells) as compared to young rats. MRP4 expression increased 3.6-fold in senescent compared to young rat myocardium (P = 0.012, n = 8 rats per group). In senescent rats, inhibition of MRP4 by MK571 restored the positive inotropic effect of isoproterenol in vivo (143 ± 11%, n = 8 rats). In vitro in senescent cardiomyocytes pretreated with MK571, both sarcomere shortening (161 ± 45% vs. 129 ± 37%, P = 0.007, n = 41 cells per group) and calcium transient amplitude (132 ± 25% vs. 113 ± 27%, P = 0.007) increased significantly. MRP4 overexpression contributes to the reduction of the positive inotropic response to β-adrenoceptor stimulation in the senescent heart.

  4. 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

  5. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Are dinucleoside monophosphates relevant models for the study of DNA intrastrand cross-link lesions? The example of g[8-5m]T.

    Science.gov (United States)

    Garrec, Julian; Dumont, Elise

    2014-07-21

    Oxidatively generated tandem lesions such as G[8-5m]T pose a potent threat to genome integrity. Direct experimental studies of the kinetics and thermodynamics of a specific lesion within DNA are very challenging, mostly due to the variety of products that can be formed in oxidative conditions. Dinucleoside monophosphates (DM) involving only the reactive nucleobases in water represent appealing alternative models on which most physical chemistry and structural techniques can be applied. However, it is not yet clear how relevant these models are. Here, we present QM/MM MD simulations of the cyclization step involved in the formation of G[8-5m]T from the guanine-thymine (GpT) DM in water, with the aim of comparing our results to our previous investigation of the same reaction in DNA ( Garrec , J. , Patel , C. , Rothlisberger , U. , and Dumont , E. ( 2012 ) J. Am. Chem. Soc. 134 , 2111 - 2119 ). We show that, despite the different levels of preorganization of the two systems, the corresponding reactions share many energetic and structural characteristics. The main difference lies in the angle between the G and T bases, which is slightly higher in the transition state (TS) and product of the reaction in water than in the reaction in DNA. This effect is due to the Watson-Crick H-bonds, which are absent in the {GpT+water} system and restrain the relative positioning of the reactive nucleobases in DNA. However, since the lesion is accommodated easily in the DNA macromolecule, the induced energetic penalty is relatively small. The high similarity between the two reactions strongly supports the use of GpT in water as a model of the corresponding reaction in DNA.

  7. 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

    2016-07-01

    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.

  8. Visualization of drug-nucleic acid interactions at atomic resolution. I. Structure of an ethidium/dinucleoside monophosphate crystalline complex, ethidium:5-iodouridylyl(3'5')adenosine

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, C C; Jain, S C; Sobell, H M

    1977-01-01

    Ethidium forms a crystalline complex with the dinucleoside monophosphate 5-iodouridyly(3'-5')adenosine (iodoUpA). These crystals are monoclinic, space group C2, with unit cell dimensions, a = 28.45 A, b = 13.54 A, c = 34.13 A, ..beta.. = 98.6/sup 0/. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares to a residual of 0.20 on 2017 observed reflections. The asymmetric unit contains two ethidium molecules, two iodoUpA molecules and 27 water molecules, a total of 155 atoms excluding hydrogens. The two iodoUpA molecules are held together by adenine.uracil Watson--Crick-type base-pairing. Adjacent base-pairs within this paired iodoUpA structure and between neighboring iodoUpA molecules in adjoining unit cells are separated by about 6.7 A; this separation results from intercalative binding by one ethidium molecule and stacking by the other ethidium molecule above and below the base-pairs. Non-crystallographic 2-fold symmetry is utilized in this model drug--nucleic acid interaction, the intercalated ethidium molecule being oriented such that its phenyl and ethyl groups lie in the narrow groove of the miniature nucleic acid double-helix. Base-pairs within the paired nucleotide units are related by a twist of 8/sup 0/. The magnitude of this angular twist is related to conformational changes in the sugar--phosphate chains that accompany drug intercalation. These changes partly reflect the differences in ribose sugar ring puckering that are observed. Additional small but systematic changes occur in torsional angles that involve the phosphodiester linkages and the C4'--C5' bond. Solution studies have indicated a marked sequence-specific binding preference in ethidium--dinucleotide interactions, and a probable structural explanation for this is provided by this study.

  9. Enhanced Bacterial α(2,6-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate.

    Directory of Open Access Journals (Sweden)

    Ji-Yeon Kang

    Full Text Available Bacterial α(2,6-sialyltransferases (STs from Photobacterium damsela, Photobacterium sp. JT-ISH-224, and P. leiognathi JT-SHIZ-145 were recombinantly expressed in Escherichia coli and their ST activities were compared directly using a galactosylated bi-antennary N-glycan as an acceptor substrate. In all ST reactions, there was an increase of sialylated glycans at shorter reaction times and later a decrease in prolonged reactions, which is related with the inherent sialidase activities of bacterial STs. These sialidase activities are greatly increased by free cytidine monophosphate (CMP generated from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac during the ST reactions. The decrease of sialylated glycans in prolonged ST reaction was prevented through an inhibition of sialidase activity by simple treatment of alkaline phosphatase (AP, which dephosphorylates CMP to cytidine. Through supplemental additions of AP and CMP-Neu5Ac to the reaction using the recombinant α(2,6-ST from P. leiognathi JT-SHIZ-145 (P145-ST, the content of bi-sialylated N-glycan increased up to ~98% without any decrease in prolonged reactions. This optimized P145-ST reaction was applied successfully for α(2,6-sialylation of asialofetuin, and this resulted in a large increase in the populations of multi-sialylated N-glycans compared with the reaction without addition of AP and CMP-Neu5Ac. These results suggest that the optimized reaction using the recombinant P145-ST readily expressed from E. coli has a promise for economic glycan synthesis and glyco-conjugate remodeling.

  10. Effect of two non-synonymous ecto-5'-nucleotidase variants on the genetic architecture of inosine 5'-monophosphate (IMP) and its degradation products in Japanese Black beef.

    Science.gov (United States)

    Uemoto, Yoshinobu; Ohtake, Tsuyoshi; Sasago, Nanae; Takeda, Masayuki; Abe, Tsuyoshi; Sakuma, Hironori; Kojima, Takatoshi; Sasaki, Shinji

    2017-11-13

    Umami is a Japanese term for the fifth basic taste and is an important sensory property of beef palatability. Inosine 5'-monophosphate (IMP) contributes to umami taste in beef. Thus, the overall change in concentration of IMP and its degradation products can potentially affect the beef palatability. In this study, we investigated the genetic architecture of IMP and its degradation products in Japanese Black beef. First, we performed genome-wide association study (GWAS), candidate gene analysis, and functional analysis to detect the causal variants that affect IMP, inosine, and hypoxanthine. Second, we evaluated the allele frequencies in the different breeds, the contribution of genetic variance, and the effect on other economical traits using the detected variants. A total of 574 Japanese Black cattle were genotyped using the Illumina BovineSNP50 BeadChip and were then used for GWAS. The results of GWAS showed that the genome-wide significant single nucleotide polymorphisms (SNPs) on BTA9 were detected for IMP, inosine, and hypoxanthine. The ecto-5'-nucleotidase (NT5E) gene, which encodes the enzyme NT5E for the extracellular degradation of IMP to inosine, was located near the significant region on BTA9. The results of candidate gene analysis and functional analysis showed that two non-synonymous SNPs (c.1318C > T and c.1475 T > A) in NT5E affected the amount of IMP and its degradation products in beef by regulating the enzymatic activity of NT5E. The Q haplotype showed a positive effect on IMP and a negative effect on the enzymatic activity of NT5E in IMP degradation. The two SNPs were under perfect linkage disequilibrium in five different breeds, and different haplotype frequencies were seen among breeds. The two SNPs contribute to about half of the total genetic variance in IMP, and the results of genetic relationship between IMP and its degradation products showed that NT5E affected the overall concentration balance of IMP and its degradation products

  11. Regulation of cessation of respiration and killing by cyclic 3',5'-adenosine monophosphate and its receptor protein after far-ultraviolet irradiation of Escherichia coli

    International Nuclear Information System (INIS)

    Swenson, P.A.; Schenley, R.L.; Joshi, J.G.

    1978-01-01

    When Escherichia coli B/r cultures are irradiated with ultraviolet light (UV) (254 nm), those cells that are killed stop respiring by 60 min after irradiation. Post-UV treatment with cyclic adenosine 3',5'-adenosine monophosphate (cAMP) causes more cells to stop respiring and to die. We have studied these effects at a UV fluence of 52 I/m 2 in a a wild-type E. coli K 12 strain and in mutants defective in cAMP metabolism. Strain CA 8,000 has crp + and cya + genes for the cAMP receptor protein (CRP) (required for transcription of operons regulated by cAMP) and for adenylate cyclase, respectively; CA 7901 is crp - ; and CA 8306 is a cya deletion (Δ). The wild-type culture showed a small transient cessation of respiration, and addition of cAMP caused cessation to be nearly complete. The crp - culture showed no evidence of cessation of respiration, and cAMP had no effect. The Δ cya mutant also showed no cessation of respiration, but cAMP (5 mM) caused as complete inhibition as in the wild type. cAMP caused a 10-fold loss in viability of UV-irradiated wild-type and Δ cya liquid cultures but had no effect on the cpr - culture. Respiration and viability changes were also studied in a double mutant, CA8404 Δ cya crp*, which has an altered CRP that is, with respect to the lac operon, independent of cAMP. The respiration response to UV was similar to that of the wild-type culture, and both respiration and viability of cells in liquid culture were sensitive to cAMP. The survival data, obtained by plating immediately after irradiation, show the wild type, Δ cya strains, and Δ cya crp* to be equally sensitive and the crp - strain to be more resistant. We conclude that cessation of respiration and cell killing after UV irradiation are regulated by cAMP and the CRP. (orig.) [de

  12. Roles of p300 and cyclic adenosine monophosphate response element binding protein in high glucose-induced hypoxia-inducible factor 1α inactivation under hypoxic conditions.

    Science.gov (United States)

    Ding, Lingtao; Yang, Minlie; Zhao, Tianlan; Lv, Guozhong

    2017-05-01

    Given the high prevalence of diabetes and burn injuries worldwide, it is essential to dissect the underlying mechanism of delayed burn wound healing in diabetes patients, especially the high glucose-induced hypoxia-inducible factor 1 (HIF-1)-mediated transcription defects. Human umbilical vein endothelial cells were cultured with low or high concentrations of glucose. HIF-1α-induced vascular endothelial growth factor (VEGF) transcription was measured by luciferase assay. Immunofluorescence staining was carried out to visualize cyclic adenosine monophosphate response element binding protein (CREB) localization. Immunoprecipitation was carried out to characterize the association between HIF-1α/p300/CREB. To test whether p300, CREB or p300+CREB co-overexpression was sufficient to rescue the HIF-1-mediated transcription defect after high glucose exposure, p300, CREB or p300+CREB co-overexpression were engineered, and VEGF expression was quantified. Finally, in vitro angiogenesis assay was carried out to test whether the high glucose-induced angiogenesis defect is rescuable by p300 and CREB co-overexpression. Chronic high glucose treatment resulted in impaired HIF-1-induced VEGF transcription and CREB exclusion from the nucleus. P300 or CREB overexpression alone cannot rescue high glucose-induced HIF-1α transcription defects. In contrast, co-overexpression of p300 and CREB dramatically ameliorated high glucose-induced impairment of HIF-1-mediated VEGF transcription, as well as in vitro angiogenesis. Finally, we showed that co-overexpression of p300 and CREB rectifies the dissociation of HIF-1α-p300-CREB protein complex in chronic high glucose-treated cells. Both p300 and CREB are required for the function integrity of HIF-1α transcription machinery and subsequent angiogenesis, suggesting future studies to improve burn wound healing might be directed to optimization of the interaction between p300, CREB and HIF-1α. © 2016 The Authors. Journal of Diabetes

  13. Pharmacological evidence for the involvement of the NMDA receptor and nitric oxide pathway in the antidepressant-like effect of lamotrigine in the mouse forced swimming test.

    Science.gov (United States)

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

    2016-08-01

    Lamotrigine is an anticonvulsant agent that shows clinical antidepressant properties. The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) synthesis in possible antidepressant-like effect of lamotrigine in forced swimming test (FST) in mice. Intraperitoneal administration of lamotrigine (10mg/kg) decreased the immobility time in the FST (P<0.01) without any effect on locomotor activity in the open-field test (OFT), while higher dose of lamotrigine (30mg/kg) reduced the immobility time in the FST (P<0.001) as well as the number of crossings in the OFT. Pretreatment of animals with NMDA (75mg/kg), l-arginine (750mg/kg, a substrate for nitric oxide synthase [NOS]) or sildenafil (5mg/kg, a phosphodiesterase [PDE] 5 inhibitor) reversed the antidepressant-like effect of lamotrigine (10mg/kg) in the FST. Injection of l-nitroarginine methyl ester (l-NAME, 10mg/kg, a non-specific NOS inhibitor), 7-nitroindazole (30mg/kg, a neuronal NOS inhibitor), methylene blue (20mg/kg, an inhibitor of both NOS and soluble guanylate cyclase [sGC]), or MK-801 (0.05mg/kg), ketamine (1mg/kg), and magnesium sulfate (10mg/kg) as NMDA receptor antagonists in combination with a sub-effective dose of lamotrigine (5mg/kg) diminished the immobility time of animals in the FST compared with either drug alone. None of the drugs produced significant effects on the locomotor activity in the OFT. Based on our findings, it is suggested that the antidepressant-like effect of lamotrigine might mediated through inhibition of either NMDA receptors or NO-cGMP synthesis. Copyright © 2016. Published by Elsevier Masson SAS.

  14. [Sexual dysfunctions linked with prostatic diseases].

    Science.gov (United States)

    Rouprêt, M; Seisen, T; De La Taille, A; Desgrandchamps, F

    2012-06-01

    The lower urinary tract symptoms (LUTS) related to benign prostatic hyperplasia (BPH) and the treatment of prostate cancer (PCa) are linked to erectile dysfunction (ED). The objective of this work was to evaluate the influence of prostatic diseases on ED. Data on the influence of BPH and PCa on ED have been explored in Medline and Embase using the MeSH keywords: benign prostatic hyperplasia, prostate cancer, prostatectomy, external beam radiotherapy; androgen deprivation therapy; erectile dysfunction. The articles were selected based on their methodology, relevance, date and language of publication. The rate of ED in patients with BPH ranged from 30 to 70 %. The LUTS were an independent risk factor of ED. The pathophysiology linking BPH to ED has not been elucidated but seems to involve the path of Nitric Oxide - cyclic Guanosine Monophosphate (cGMP-No.), the RhoA - Rho - Kinase (ROCK) signal, the sympathetic autonomic nervous system and pelvic atherosclerosis. The rate of ED after radical prostatectomy (RP) ranged from 60 to 89 %. The bilateral preservation of neurovascular bundels improved these results. Risk factors of ED after RP were age, PSA levels, pretreatment erectile function and surgical technique. The rate of ED after prostate external beam radiotherapy ranged from 6 to 84 %. Risk factors of ED after external beam radiotherapy were age, pretreatment erectile function and association of androgen deprivation therapy. The rate of ED with androgen deprivation therapy was 85 %. Risk factors of ED with androgen deprivation therapy were age > 70 years, diabetes and pretreatment erectile function. Intermittent androgen deprivation therapy was associated with better results on erectile function than continue androgen deprivation therapy. ED is responsible for a decrease of elderly patients life quality already affected by urinary symptoms and prostate disease progression. The development of drugs effective on both ED and BPH or PCa symptoms is then full of

  15. Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Percival, Justin M; Whitehead, Nicholas P; Adams, Marvin E; Adamo, Candace M; Beavo, Joseph A; Froehner, Stanley C

    2012-09-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO

  16. A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP

    Directory of Open Access Journals (Sweden)

    Xin Jia

    2014-12-01

    Full Text Available A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s and interplay of cNMP signalling pathways.

  17. A facile and sensitive method for quantification of cyclic nucleotide monophosphates in mammalian organs: basal levels of eight cNMPs and identification of 2',3'-cIMP.

    Science.gov (United States)

    Jia, Xin; Fontaine, Benjamin M; Strobel, Fred; Weinert, Emily E

    2014-12-12

    A sensitive, versatile and economical method to extract and quantify cyclic nucleotide monophosphates (cNMPs) using LC-MS/MS, including both 3',5'-cNMPs and 2',3'-cNMPs, in mammalian tissues and cellular systems has been developed. Problems, such as matrix effects from complex biological samples, are addressed and have been optimized. This protocol allows for comparison of multiple cNMPs in the same system and was used to examine the relationship between tissue levels of cNMPs in a panel of rat organs. In addition, the study reports the first identification and quantification of 2',3'-cIMP. The developed method will allow for quantification of cNMPs levels in cells and tissues with varying disease states, which will provide insight into the role(s) and interplay of cNMP signalling pathways.

  18. 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.

  19. The fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms improves insulin resistance and hepatic lipid accumulation by modulation of liver adenosine monophosphate-activated protein kinase activity and lipogenic gene expression in high-fat diet-fed obese mice.

    Science.gov (United States)

    Saito, Tetsuo; Nishida, Miyako; Saito, Masafumi; Tanabe, Akari; Eitsuka, Takahiro; Yuan, Shi-Hua; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-10-01

    Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate-activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD)-induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism-associated gene expression. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)β(1).

    Science.gov (United States)

    Beste, Kerstin Y; Seifert, Roland

    2013-02-01

    Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate are second messengers that regulate multiple physiological functions. The existence of additional cyclic nucleotides in mammalian cells was postulated many years ago, but technical problems hampered development of the field. Using highly specific and sensitive mass spectrometry methods, soluble guanylyl cyclase has recently been shown to catalyze the formation of several cyclic nucleotides in vitro. This minireview discusses the broad substrate-specificity of soluble guanylyl cyclase and the possible second messenger roles of cyclic nucleotides other than adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. We hope that this article stimulates productive and critical research in an area that has been neglected for many years.

  1. Inhibition of excitatory synaptic transmission in the trigeminal motor nucleus by the nitric oxide-cyclic GMP signaling pathway.

    Science.gov (United States)

    Pose, Inés; Silveira, Valentina; Morales, Francisco R

    2011-06-01

    Nitric oxide (NO) and cyclic GMP (cGMP) suppressed glutamatergic synaptic transmission to trigeminal motoneurons in brain stem slices of neonatal rats. Histological studies showed guanylate cyclase (GC) containing fibers in the trigeminal motor pool. Glutamatergic excitatory postsynaptic currents (EPSCs) were recorded from neonatal trigeminal motoneurons in response to stimulation of the supratrigeminal nucleus (SuV). The NO donors DETA/NONOate (DETA/NO), at a concentration which released 275.1 nM of NO, and Spermine/NONOate (Sper/NO) reduced the amplitude of the EPSC to 52.7±0.6% and 60.1±10.8% of control values, respectively. These actions were not blocked by the GC inhibitors, ODQ or NS-2028. However, in the presence of YC-1 or BAY41-2272, modulators of GC that act as NO sensitizers, lower and otherwise ineffective concentrations of DETA/NO induced a reduction of the EPSC to 60.6±5.2%. Moreover, NO effects were mimicked by 8BrcGMP and by Zaprinast, an inhibitor of Phosphodiesterase 5. Glutamatergic currents evoked by exogenous glutamate were not reduced by DETA/NO nor 8BrcGMP. Paired-pulse facilitation was increased by NO donors. Under "minimal stimulation" conditions NO donors and cGMP increased the failure rate of evoked EPSCs. Protein kinase inhibitors antagonized cGMP effects. The results suggest that NO, through the synthesis of cGMP, presynaptically inhibits glutamatergic synaptic transmission on trigeminal motoneurons. We propose that NO has complex actions on motor pools; specific studies are needed to elucidate their physiological significance in the behaving animal. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

    Directory of Open Access Journals (Sweden)

    Yingshuai Zhao

    2017-05-01

    Full Text Available Valsartan (VAL, an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO. In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs. Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L, VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L, and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L groups. The NO content in the VAL-treated HUVEC line (EA.hy926 was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05 and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

  3. Sliding charge density wave in the monophosphate tungsten bronze (PO2)4(WO3)2m with alternate stacking of m=4 and m=6 WO3 layers

    International Nuclear Information System (INIS)

    Foury-Leylekian, P.; Sandre, E.; Ravy, S.; Pouget, J.-P.; Elkaim, E.; Roussel, P.; Groult, D.; Labbe, Ph.

    2002-01-01

    The monophosphate tungsten bronzes (PO 2 ) 4 (WO 3 ) 2m form family of two-dimensional metals which exhibit charge density wave (CDW) instabilities. These materials are generally built by the regular stacking of (a,b) layers in which chains made of segments of m WO 6 octahedra directed along the a and a±b directions are delimited. Their electronic structure thus originates from quasi-one-dimensional (1D) bands located on these chains. As a consequence their Fermi surface (FS) exhibits large flat portions whose nesting gives rise to successive CDW instabilities. Here we present a structural study of the CDW instability of the (PO 2 ) 4 (WO 3 ) 10 member formed by the alternate stacking of layers built with segments of m=4 and m=6 WO 6 octahedra. Its ab initio electronic structure calculation shows that the FS of this member exhibits large flat portions which can be extremely well nested. Its best nesting wave vector accounts for the modulation wave vector stabilized by the CDW transition which occurs at 156 K. Because of the regular stacking of layers of different m values the FS is slightly split. The unusual thermal dependence of the x-ray satellite intensity provides evidence that the two types of layers become modulated at different temperature. This also leads to a slight thermal sliding of the CDW-nesting modulation wave vector, which can be accounted for within the framework of a Landau-Ginzburg theory. In addition, the observation of a global hysteresis in the thermal cycling of the satellite intensity, as well as the degradation of the interlayer order upon cooling, suggest the formation of a disordered lattice of dilute solitons. Such solitons allow to accommodate the charge transferred between the two types of layer. Finally the relevance of local charge transfers, at intergrowth defects, for example, to create pinned discommensurations that break the CDW coherence is emphasized in this whole family of bronzes

  4. Hepatic oxidative stress in ovariectomized transgenic mice expressing the hepatitis C virus polyprotein is augmented through suppression of adenosine monophosphate-activated protein kinase/proliferator-activated receptor gamma co-activator 1 alpha signaling.

    Science.gov (United States)

    Tomiyama, Yasuyuki; Nishina, Sohji; Hara, Yuichi; Kawase, Tomoya; Hino, Keisuke

    2014-10-01

    Oxidative stress plays an important role in hepatocarcinogenesis of hepatitis C virus (HCV)-related chronic liver diseases. Despite the evidence of an increased proportion of females among elderly patients with HCV-related hepatocellular carcinoma (HCC), it remains unknown whether HCV augments hepatic oxidative stress in postmenopausal women. The aim of this study was to determine whether oxidative stress was augmented in ovariectomized (OVX) transgenic mice expressing the HCV polyprotein and to investigate its underlying mechanisms. OVX and sham-operated female transgenic mice expressing the HCV polyprotein and non-transgenic littermates were assessed for the production of reactive oxygen species (ROS), expression of inflammatory cytokines and antioxidant potential in the liver. Compared with OVX non-transgenic mice, OVX transgenic mice showed marked hepatic steatosis and ROS production without increased induction of inflammatory cytokines, but there was no increase in ROS-detoxifying enzymes such as superoxide dismutase 2 and glutathione peroxidase 1. In accordance with these results, OVX transgenic mice showed less activation of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), which is required for the induction of ROS-detoxifying enzymes, and no activation of adenosine monophosphate-activated protein kinase-α (AMPKα), which regulates the activity of PGC-1α. Our study demonstrated that hepatic oxidative stress was augmented in OVX transgenic mice expressing the HCV polyprotein by attenuation of antioxidant potential through inhibition of AMPK/PGC-1α signaling. These results may account in part for the mechanisms by which HCV-infected women are at high risk for HCC development when some period has passed after menopause. © 2013 The Japan Society of Hepatology.

  5. An Examination of the Role of L-Glutamate and Inosine 5'-Monophosphate in Hedonic Taste-Guided Behavior by Mice Lacking the T1R1 + T1R3 Receptor.

    Science.gov (United States)

    Blonde, Ginger D; Spector, Alan C

    2017-06-01

    The heterodimeric T1R1 + T1R3 receptor is considered critical for normal signaling of L-glutamate and 5'-ribonucleotides in the oral cavity. However, some taste-guided responsiveness remains in mice lacking one subunit of the receptor, suggesting that other receptors are sufficient to support some behaviors. Here, mice lacking both receptor subunits (KO) and wild-type (WT, both n = 13) mice were tested in a battery of behavioral tests. Mice were trained and tested in gustometers with a concentration series of Maltrin-580, a maltodextrin, in a brief-access test (10-s trials) as a positive control. Similar tests followed with monosodium glutamate (MSG) with and without the ribonucleotide inosine 5'-monophosphate (IMP), but always in the presence of the epithelial sodium channel blocker amiloride (A). Brief-access tests were repeated following short-term (30-min) and long-term (48-h) exposures to MSG + A + IMP and were also conducted with sodium gluconate replacing MSG. Finally, progressive ratio tests were conducted with Maltrin-580 or MSG + A + IMP, to assess appetitive behavior while minimizing satiation. Overall, MSG generated little concentration-dependent responding in either food-restricted WT or KO mice, even in combination with IMP. However, KO mice licked less to the amino acid stimuli, a measure of consummatory behavior in the brief-access tests. In contrast, both groups initiated a similar number of trials and had a similar breakpoint in the progressive ratio task, both measures of appetitive (approach) behavior. Collectively, these results suggest that while the T1R1 + T1R3 receptor is necessary for consummatory responding to MSG (+IMP), other receptors are sufficient to maintain appetitive responding to this "umami" stimulus complex in food-restricted mice. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. New Inosine and Guanosine Analogs as Inhibitors of Parasitic Infections.

    Science.gov (United States)

    1985-11-30

    infections. Although chloroquine (CQ) is generally considered to be one of the most fascinating, useful and versatile drugs developed during the modern...ribonucleosides are of particular interest since these nucleosides may be looked upon as aza- analogues of formycin B. The parent s-triazolo[3,4-f]-as...hexopyranosyl)adenine indicate slightly altered glycon. This type of nucleoside analogues could mimic either as ribonucleo- sides or as 2

  7. Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

    Science.gov (United States)

    De Silva, Matharage S I; Dayton, Adam W; Rhoten, Lance R; Mallett, John W; Reese, Jared C; Squires, Mathieu D; Dalley, Andrew P; Porter, James P; Judd, Allan M

    2018-06-01

    In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

    Science.gov (United States)

    Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika

    2016-12-30

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. 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

  10. Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

    Science.gov (United States)

    Fujita, Akiko; Sato, Chihiro; Münster-Kühnel, Anja-K; Gerardy-Schahn, Rita; Kitajima, Ken

    2005-02-01

    A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay

  11. Osmium(VI) complexes as a new class of potential anti-cancer agents.

    Science.gov (United States)

    Ni, Wen-Xiu; Man, Wai-Lun; Cheung, Myra Ting-Wai; Sun, Raymond Wai-Yin; Shu, Yuan-Lan; Lam, Yun-Wah; Che, Chi-Ming; Lau, Tai-Chu

    2011-02-21

    A nitridoosmium(VI) complex [Os(VI)(N)(sap)(OH(2))Cl] (H(2)sap = N-salicylidene-2-aminophenol) displays prominent in vitro and in vivo anti-cancer properties, induces S- and G2/M-phase arrest and forms a stable adduct with dianionic 5'-guanosine monophosphate.

  12. Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration

    NARCIS (Netherlands)

    Ivanova, Krassimira; Zadeh, Nahid Hamidi; Block, Ingrid; Das, Pranab K.; Gerzer, Rupert

    2004-01-01

    Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic

  13. Nitric Oxide Binds to and Modulates the Activity of a Pollen Specific Arabidopsis Diacylglycerol Kinase

    KAUST Repository

    Wong, Aloysius Tze

    2014-01-01

    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

  14. Bacterial Biofilm Control by Perturbation of Bacterial Signaling Processes

    DEFF Research Database (Denmark)

    Jakobsen, Tim Holm; Tolker-Nielsen, Tim; Givskov, Michael

    2017-01-01

    -dimeric guanosine monophosphate (cyclic-di-GMP) have gained a lot of attention over the last two decades. More recently, regulatory processes governed by two component regulatory systems and small non-coding RNAs have been increasingly investigated. Here, we review novel findings and potentials of using small...

  15. Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

    2015-01-01

    The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One...

  16. Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

    KAUST Repository

    Marondedze, Claudius; Turek, Ilona; Parrott, Brian Jonathan; Thomas, Ludivine; Jankovic, Boris R.; Lilley, Kathryn S; Gehring, Christoph A

    2013-01-01

    molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by c

  17. Transient Kinetics of a cGMP-dependent cGMP-specific Phosphodiesterase from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van

    1984-01-01

    Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in ~30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme

  18. The Beneficial Effect of Fesoterodine, a Competitive Muscarinic Receptor Antagonist on Erectile Dysfunction in Streptozotocin-induced Diabetic Rats.

    Science.gov (United States)

    Yilmaz-Oral, Didem; Bayatli, Nur; Gur, Serap

    2017-09-01

    To investigate the possible role of fesoterodine (a competitive muscarinic receptor antagonist) on erectile dysfunction in streptozotocin-induced diabetic rats. A total of 16 adult male Sprague-Dawley rats were equally divided into control and diabetic groups. Diabetes was induced by a single intravenous injection of streptozotocin (25-35 mg/kg). In vivo erectile responses were evaluated by the stimulation of cavernosal nerves, and measurements were repeated after the intracavernosal injection of fesoterodine (1 µM) in rats. The relaxation responses to fesoterodine were examined via incubation with various inhibitors. The relaxant responses of corpus cavernosum (CC) strips were observed in the presence or the absence of fesoterodine (10 µM). Intracavernous administration of fesoterodine restored in vivo erectile response at 5.0- and 7.5-V levels, except for 2.5 V in diabetic rats. Basal intracavernosal pressure (5.4 ± 0.9 mm Hg) in diabetic rats was markedly increased after injection of fesoterodine (33.9 ± 7.9 mm Hg, P <.001). In bath studies, fesoterodine resulted in a relaxation of CC in a concentration-dependent manner, which was reduced in diabetic rats. Nifedipine (l-type Ca 2+ channel blocker) inhibited maximum fesoterodine-induced relaxation by 58%. The nonselective K + channel blocker tetraethylammonium and glibenclamide incubation did not change the relaxant response to fesoterodine. The relaxant responses to acetylcholine (10 µM), electrical field stimulation (10 Hz), and sodium nitroprusside (0.01 µM) in diabetic rats were increased after incubation with fesoterodine (10 µM). Fesoterodine improved erectile function and relaxation of isolated strips of rat CC. The underlying mechanism of fesoterodine is likely due to the blocking of l-type calcium channels independent of the nitric oxide-cyclic guanosine monophosphate pathway. Further investigations are warranted to fully elucidate the restorative effects of

  19. Antidepressant effect of pramipexole in mice forced swimming test: A cross talk between dopamine receptor and NMDA/nitric oxide/cGMP pathway.

    Science.gov (United States)

    Ostadhadi, Sattar; Imran Khan, Muhammad; Norouzi-Javidan, Abbas; Dehpour, Ahmad-Reza

    2016-07-01

    Pramipexole is a dopamine D2 receptor agonist indicated for treating Parkinson disorder. This study was aimed to investigate the effect of pramipexole in forced swimming test (FST) in mice and the possible involvement of activation of D2 receptors and inhibition of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) on this effect. Intraperitoneal administration of pramipexole (1-3mg/kg) reduced the immobility time in the FST similar to fluoxetine (20mg/kg, i.p.). This effect of pramipexole (1mg/kg, i.p.) was ceased when mice were pretreated with haloperidol (0.15mg/kg, i.p,) and sulpiride (5mg/kg, i.p) as D2 receptor antagonists, NMDA (75mg/kg,i.p.), l-arginine (750mg/kg, i.p., a substrate for nitric oxide synthase) or sildenafil (5mg/kg, i.p., a phosphodiesterase 5 inhibitor). The administration of MK-801 (0.05mg/kg, i.p., a NMDA receptor antagonist) l-NG-Nitro arginine methyl ester (l-NAME, 10mg/kg, i.p., a non-specific nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (30mg/kg, i.p., a neuronal NOS inhibitor) and methylene blue (10mg/kg, i.p.), an inhibitor of both NOS and soluble guanylyl cyclase (sGC) in combination with the sub-effective dose of pramipexole (0.3mg/kg, i.p.) reduced the immobility. Altogether, our data suggest that the antidepressant-like effect of pramipexole is dependent on the activation of D2 receptor and inhibition of either NMDA receptors and/or NO-cGMP synthesis. These results contribute to the understanding of the mechanisms underlying the antidepressant-like effect of pramipexole and reinforce the role of D2 receptors, NMDA receptors and l-arginine-NO-GMP pathway in the antidepressant mechanism of this agent. Copyright © 2016. Published by Elsevier Masson SAS.

  20. Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide-cyclic GMP pathway in mice.

    Science.gov (United States)

    da Silva, F H; Pereira, M N; Franco-Penteado, C F; De Nucci, G; Antunes, E; Claudino, M A

    2013-01-01

    Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg(-1) per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration-responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8-32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction.

  1. Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1beta.

    Science.gov (United States)

    Palmi, Mitri; Meini, Antonella

    2002-04-01

    Interleukin-1beta (IL-1beta) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca2+ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1beta and other pyrogens. Data from different labs indicate that Ca2+ and Na+ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca2+ and PGE2 concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1beta-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204-212) peptide counteract IL-1beta-induced fever and abolish changes in Ca2+ and PGE2 concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca2+ mobilization in response to IL-1beta and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP3)-sensitive pools are the main source of the mobilized Ca2+. It is concluded that the NO/cGMP/Ca2+ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1beta.

  2. The role of cGMP signalling in regulating life cycle progression of Plasmodium.

    OpenAIRE

    Hopp, CS; Bowyer, PW; Baker, DA

    2012-01-01

    The 3′-5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regu...

  3. cGMP Signaling in the Cardiovascular System—The Role of Compartmentation and Its Live Cell Imaging

    Science.gov (United States)

    Bork, Nadja I.; Nikolaev, Viacheslav O.

    2018-01-01

    The ubiquitous second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) regulates multiple physiologic processes in the cardiovascular system. Its intracellular effects are mediated by stringently controlled subcellular microdomains. In this review, we will illustrate the current techniques available for real-time cGMP measurements with a specific focus on live cell imaging methods. We will also discuss currently accepted and emerging mechanisms of cGMP compartmentation in the cardiovascular system. PMID:29534460

  4. Effects of cyclic-nucleotide derivatives on the growth of human colonic carcinoma xenografts and on cell production in the rat colonic crypt epithelium.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1981-08-01

    Previous studies have shown that various amine hormones are able to influence the growth rate of human colorectal carcinomas propagated as xenografts in immune-deprived mice, and it is now well known that the effects of many amine and other hormones are mediated by cyclic nucleotides, acting as second messengers within cells. In the present study the influence of various derivatives of cyclic adenosine monophosphate and cyclic guanosine monophosphate on the growth of two different lines of colorectal cancer growing in immune-deprived mice, and on the cell production rate in the colonic crypt epithelium of the rat, was assessed. Growth of each tumour line, as well as crypt-cell production, was suppressed by treatment wit N6O2' dibutyryl and N6 monobutyryl derivatives of cyclic adenosine monophosphate. Dibutyryl cyclic guanosine monophosphate, on the other hand, was found to promote the growth of Tumour HXK4 and to promote crypt cell production, but to have no significant effect on Tumour HXM2.

  5. Detection of DNA nucleotides on pretreated boron doped diamond electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Garbellini, Gustavo S.; Uliana, Carolina V.; Yamanaka, Hideko [UNESP, Araraquara, SP (Brazil). Inst. de Quimica

    2011-07-01

    The individual detection and equimolar mixture of DNA nucleotides guanosine monophosphate (GMP), adenosine monophosphate (AMP), thymidine (TMP) and cytidine (CMP) 5'-monophosphate using square wave voltammetry was performed on boron doped diamond (BDD) electrodes cathodically (Red-DDB) and anodically (Oxi-DDB) pretreated. The oxidation of individual DNA nucleotides was more sensitive on Oxi-BDD electrode. In a simultaneous detection of nucleotides, the responses of GMP, AMP, TMP and CMP were very adequate on both treated electrodes. Particularly, more sensitive and separate peaks for TMP and CMP on Oxi-BDD and Red-BDD electrodes, respectively, were observed after deconvolution procedure. The detection of nucleotides in aqueous solutions will certainly contribute for genotoxic evaluation of substances and hybridization reactions by immobilizing ss or ds-DNA on BDD surface. (author)

  6. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    2016-12-01

    Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. ... have shown that the stress associated with cold temperature ..... vation by cyclic-AMP-dependent protein kinase, studied using.

  7. Adenosine monophosphate-activated protein kinase from the mud ...

    Indian Academy of Sciences (India)

    CHENCUI HUANG

    would lead to disturbance of aerobic metabolism and reduc- tion of ATP ... the effects of heat temperature stress on the expression of. AMPK have ..... both a respiratory and osmoregulation organ, gill directly in ..... response to salinity stress.

  8. Isosteric phosphonate pyrrolidine-based dinucleoside monophosphate analogues

    Czech Academy of Sciences Publication Activity Database

    Vaněk, Václav; Buděšínský, Miloš; Kavenová, Ivana; Rinnová, Markéta; Rosenberg, Ivan

    2003-01-01

    Roč. 22, 5/8 (2003), s. 1065-1067 ISSN 1525-7770. [International Roundtable Nucleosides, Nucleotides and Nucleic Acids /15./. Leuven, 10.09.2002-14.09.2002] R&D Projects: GA ČR GA203/01/1166; GA AV ČR IAA4055101 Institutional research plan: CEZ:AV0Z4055905 Keywords : pyrrolidine-based phosphonate nucleotides * ApA analogues * triplex Subject RIV: CC - Organic Chemistry Impact factor: 0.813, year: 2003

  9. Substrate Binding Induces Domain Movements in Orotidine 5'-Monophosphate Decarboxylase

    DEFF Research Database (Denmark)

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

    2002-01-01

    ); here we present the 2.5 Å structure of the uncomplexed apo enzyme, determined from twinned crystals. A structural analysis and comparison of the two structures of the E. coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12° around a hinge...

  10. Persistence Increases in the Absence of the Alarmone Guanosine Tetraphosphate by Reducing Cell Growth

    Science.gov (United States)

    2016-08-16

    Pennsylvania, Pennsylvania State University, University Park, 16802- 4400, USA. 2Department of Biochemistry and Molecular Biology, Pennsylvania State...of Toxin- Antitoxin Activity. Cell 154, 1140–1150 (2013). 26. Osbourne, D. O., Soo, V. W. C., Konieczny, I. & Wood, T. K. Polyphosphate, cyclic AMP

  11. Elevated levels of plasma phenylalanine in schizophrenia: a guanosine triphosphate cyclohydrolase-1 metabolic pathway abnormality?

    Directory of Open Access Journals (Sweden)

    Olaoluwa Okusaga

    Full Text Available BACKGROUND: Phenylalanine and tyrosine are precursor amino acids required for the synthesis of dopamine, the main neurotransmitter implicated in the neurobiology of schizophrenia. Inflammation, increasingly implicated in schizophrenia, can impair the function of the enzyme Phenylalanine hydroxylase (PAH; which catalyzes the conversion of phenylalanine to tyrosine and thus lead to elevated phenylalanine levels and reduced tyrosine levels. This study aimed to compare phenylalanine, tyrosine, and their ratio (a proxy for PAH function in a relatively large sample of schizophrenia patients and healthy controls. METHODS: We measured non-fasting plasma phenylalanine and tyrosine in 950 schizophrenia patients and 1000 healthy controls. We carried out multivariate analyses to compare log transformed phenylalanine, tyrosine, and phenylalanine:tyrosine ratio between patients and controls. RESULTS: Compared to controls, schizophrenia patients had higher phenylalanine (p<0.0001 and phenylalanine: tyrosine ratio (p<0.0001 but tyrosine did not differ between the two groups (p = 0.596. CONCLUSIONS: Elevated phenylalanine and phenylalanine:tyrosine ratio in the blood of schizophrenia patients have to be replicated in longitudinal studies. The results may relate to an abnormal PAH function in schizophrenia that could become a target for novel preventative and interventional approaches.

  12. Enzymatic synthesis of 5-methyluridine by transglycosylation of guanosine and thymine

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2012-05-01

    Full Text Available 5-Methyluridine (5-MU) is an intermediate in the synthesis of ß-thymidine and the antiretroviral drugs stavudine (d4T) and zidovudine (AZT)1-3. The enzymatic preparation of 5-MU involves transglycosylation4-6 and avoids the formation of unwanted...

  13. Apparent non-statistical binding in a ditopic receptor for guanosine

    NARCIS (Netherlands)

    Likhitsup, Asawin; Deeth, Robert J.; Otto, Sijbren; Marsh, Andrew

    2009-01-01

    Analysis of stepwise association constants for guests binding to more than one site in a receptor is expected to give a ratio of the first association constant to the second of about 4 : 1 on statistical grounds (since a second guest should have an equal chance of binding to a different site on the

  14. Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism.

    Science.gov (United States)

    Fukuuchi, T; Iyama, N; Yamaoka, N; Kaneko, K

    2018-04-13

    Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.

  15. Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro

    KAUST Repository

    Mulaudzi, Takalani

    2011-09-01

    While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O 2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. The role of cGMP signalling in regulating life cycle progression of Plasmodium.

    Science.gov (United States)

    Hopp, Christine S; Bowyer, Paul W; Baker, David A

    2012-08-01

    The 3'-5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is the main mediator of cGMP signalling in the malaria parasite. This article reviews the role of PKG in Plasmodium falciparum during gametogenesis and blood stage schizont rupture, as well as the role of the Plasmodium berghei orthologue in ookinete differentiation and motility, and liver stage schizont development. The current views on potential effector proteins downstream of PKG and the mechanisms that may regulate cyclic nucleotide levels are presented. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  17. Antidepressant-like properties of sildenafil in a genetic rat model of depression: Role of cholinergic cGMP-interactions

    DEFF Research Database (Denmark)

    Liebenberg, Nico; Brink, Christiaan; Brand, Linda

    2008-01-01

    Background: The N-methyl-D-aspartate (NMDA)/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway has been implicated in the neurobiology of depression. Recently we suggested a possible complex interaction between the cholinergic and NO-cGMP pathways in the antidepressant-like response....... Conclusions: Using a genetic animal model of depression, we have confirmed the antidepressant-like property of sildenafil following “unmasking” by concomitant block of muscarinic receptors. These findings hint at a novel interaction between the cGMP and cholinergic systems in depression, and suggest...

  18. In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation

    DEFF Research Database (Denmark)

    Chua, Song Lin; Hultqvist, Louise D; Yuan, Mingjun

    2015-01-01

    Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a global secondary bacterial messenger that controls the formation of drug-resistant multicellular biofilms. Lowering the intracellular c-di-GMP content can disperse biofilms, and it is proposed as a biofilm eradication strategy...... biofilms by reducing the intracellular c-di-GMP content through modulation of phosphodiesterases (PDEs). Unlike conventional protocols that demonstrate biofilm dispersal by biomass quantification, our protocols enable physiological characterization of the dispersed cells. Biomarkers of dispersed cells...

  19. Cerebral haemodynamic response or excitability is not affected by sildenafil

    DEFF Research Database (Denmark)

    Kruuse, Christina; Hansen, Adam E; Larsson, Henrik B W

    2009-01-01

    Sildenafil (Viagra), a cyclic guanosine monophosphate-degrading phosphodiesterase 5 inhibitor, induces headache and migraine. Such headache induction may be caused by an increased neuronal excitability, as no concurrent effect on cerebral arteries is found. In 13 healthy females (23+/-3 years, 70...... amplitude or latency (P100). The fMRI response to visual stimulation or hypercapnia was unchanged by sildenafil. In conclusion, sildenafil induces mild headache without potentiating a neuronal or local cerebrovascular visual response or a global cerebrovascular hypercapnic response. The implication...

  20. Effect of sildenafil on gastric emptying and postprandial frequency of antral contractions in healthy humans

    DEFF Research Database (Denmark)

    Madsen, Jan Lysgård; Søndergaard, S B; Fuglsang, Stefan

    2004-01-01

    BACKGROUND: Sildenafil is known to block phosphodiesterase type 5, which degrades nitric oxide-stimulated cyclic guanosine monophosphate, thereby relaxing smooth muscle cells in various organs. The effect of sildenafil on gastric motor function after a meal was investigated in healthy humans...... gastric emptying and postprandial frequency of antral contractions. RESULTS: The area under the curve of gastric retention versus time of liquid or solid radiolabelled marker was not changed by sildenafil intake, nor was the postprandial frequency of antral contractions affected by sildenafil. CONCLUSION......: A single dose of 50 mg sildenafil does not change gastric emptying or postprandial frequency of antral contractions in healthy volunteers....

  1. GMP reverses the facilitatory effect of glutamate on inhibitory avoidance task in rats.

    Science.gov (United States)

    Rubin, M A; Jurach, A; da Costa Júnior, E M; Lima, T T; Jiménez-Bernal, R E; Begnini, J; Souza, D O; de Mello, C F

    1996-09-02

    Previous studies have demonstrated that post-training intrahippocampal glutamate administration improves inhibitory avoidance task performance in rats. Antagonism of the agonist actions of glutamate by guanine nucleotides has been shown at the molecular and behavioural level. In the present investigation we demonstrate that intrahippocampal co-administration of GMP (guanosine 5'-monophosphate) reverses the facilitatory effect of glutamate on the inhibitory avoidance learning paradigm and inhibits [3H]glutamate binding in hippocampal synaptic plasma membranes. These results suggest that guanine nucleotides may modulate glutamate actions.

  2. Cyclic Nucleotide Signalling in Kidney Fibrosis

    Directory of Open Access Journals (Sweden)

    Elisabeth Schinner

    2015-01-01

    Full Text Available Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.

  3. Neuronal nitric oxide synthase supports Renin release during sodium restriction through inhibition of phosphodiesterase 3

    DEFF Research Database (Denmark)

    Sällström, Johan; Jensen, Boye L; Skøtt, Ole

    2010-01-01

    NOS supports renin release by cyclic guanosine monophosphate (cGMP)-mediated inhibition of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase 3 (PDE3) in juxtaglomerular (JG) cells. METHODS: The experiments were performed in conscious nNOS⁻(/)⁻ and wild types after 10 days on a low-sodium diet...... by measurements of inulin- and para-amino hippuric acid (PAH) clearances, respectively. RESULTS: The basal PRC was reduced in nNOS⁻(/)⁻ compared to the wild types. Administration of milrinone caused a more pronounced PRC increase in nNOS⁻(/)⁻, resulting in normalized renin levels, whereas PDE5 inhibition did...... not affect PRC in any genotype. The blood pressure was similar in both genotypes, and milrinone did not affect blood pressure compared to vehicle. GFR and RPF were similar at baseline and were reduced by milrinone. CONCLUSIONS: The present study provides in vivo evidence supporting the view that NO...

  4. Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba) Fruit During the Drying and Steaming Process.

    Science.gov (United States)

    Guo, Sheng; Duan, Jin-Ao; Zhang, Ying; Qian, Dawei; Tang, Yuping; Zhu, Zhenhua; Wang, Hanqing

    2015-12-12

    Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed.

  5. Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

    Science.gov (United States)

    Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

    1996-10-03

    We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

  6. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.

    Science.gov (United States)

    Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J

    2013-08-23

    Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

  7. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

    2013-02-15

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

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

    KAUST Repository

    Raji, Misjudeen; Gehring, Christoph A

    2017-01-01

    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.

  9. Studies on the influences of. gamma. -ray irradiation upon food additives, (7). Radiolysis of 5'-nucleotides due to. gamma. -ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Hamada, M [Shimonoseki Univ. of Fisheries, Yamaguchi (Japan); Gohya, Y; Ishio, S

    1981-08-01

    The effect of ..gamma..-ray irradiation on inosine-5'-monophosphate (5'-IMP) and guanosine-5'-monophosphate (5'-GMP) in aqueous solution and in ''kamaboko'' was investigated to evaluate the rate of decomposition and to elucidate the safety of the decomposed products, under the concentration of 0.025% and irradiation dose of 3.00 x 10/sup 5/ rad. Ribose-phosphate compound, inorganic phosphate and 2'-, 3'-nucleotides were ascertained when either 5'-IMP or 5'-GMP aqueous solution was irradiated. G value of 5'-IMP and 5'-GMP in aqueous solution were estimated to be 1.29 and 0.97, respectively. The radiolysis of both 5'-IMP and 5'-GMP in ''kamaboko'' was hardly proceeded.

  10. Novel Radioligands for Cyclic Nucleotide Phosphodiesterase Imaging with Positron Emission Tomography: An Update on Developments Since 2012

    Directory of Open Access Journals (Sweden)

    Susann Schröder

    2016-05-01

    Full Text Available Cyclic nucleotide phosphodiesterases (PDEs are a class of intracellular enzymes that inactivate the secondary messenger molecules, cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP. Thus, PDEs regulate the signaling cascades mediated by these cyclic nucleotides and affect fundamental intracellular processes. Pharmacological inhibition of PDE activity is a promising strategy for treatment of several diseases. However, the role of the different PDEs in related pathologies is not completely clarified yet. PDE-specific radioligands enable non-invasive visualization and quantification of these enzymes by positron emission tomography (PET in vivo and provide an important translational tool for elucidation of the relationship between altered expression of PDEs and pathophysiological effects as well as (pre-clinical evaluation of novel PDE inhibitors developed as therapeutics. Herein we present an overview of novel PDE radioligands for PET published since 2012.

  11. Plasma levels of cAMP, cGMP and CGRP in sildenafil-induced headache

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Frandsen, E; Schifter, S

    2004-01-01

    Sildenafil, a selective inhibitor of the cyclic guanosine monophosphate (cGMP) degrading phosphodiestrase 5 (PDE5), induced migraine without aura in 10 of 12 migraine patients and in healthy subjects it induced significantly more headache than placebo. The aim of the present study was to determine...... whether the pain-inducing effects of sildenafil would be reflected in plasma levels of important signalling molecules in migraine: cGMP, cyclic adenosine monophosphate (cAMP) and calcitonin gene-related peptide (CGRP). Ten healthy subjects (four women, six men) and 12 patients (12 women) suffering from...... migraine without aura were included in two separate double-blind, placebo-controlled, cross-over studies in which placebo or sildenafil 100 mg was administered orally. Plasma levels of CGRP, cAMP and cGMP were determined in blood from the antecubital vein. Despite the ability of sildenafil to induce...

  12. Active site similarity between human and Plasmodium falciparum phosphodiesterases: considerations for antimalarial drug design

    Science.gov (United States)

    Howard, Brittany L.; Thompson, Philip E.; Manallack, David T.

    2011-08-01

    The similarity between Plasmodium falciparum phosphodiesterase enzymes ( PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.

  13. 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.

  14. 3',5'-cIMP as Potential Second Messenger in the Vascular Wall.

    Science.gov (United States)

    Leung, Susan W S; Gao, Yuansheng; Vanhoutte, Paul M

    2017-01-01

    Traditionally, only the 3',5'-cyclic monophosphates of adenosine and guanosine (produced by adenylyl cyclase and guanylyl cyclase, respectively) are regarded as true "second messengers" in the vascular wall, despite the presence of other cyclic nucleotides in different tissues. Among these noncanonical cyclic nucleotides, inosine 3',5'-cyclic monophosphate (cIMP) is synthesized by soluble guanylyl cyclase in porcine coronary arteries in response to hypoxia, when the enzyme is activated by endothelium-derived nitric oxide. Its production is associated with augmentation of vascular contraction mediated by stimulation of Rho kinase. Based on these findings, cIMP appears to meet most, if not all, of the criteria required for it to be accepted as a "second messenger," at least in the vascular wall.

  15. Design, synthesis, and actions of a novel chimeric natriuretic peptide: CD-NP.

    Science.gov (United States)

    Lisy, Ondrej; Huntley, Brenda K; McCormick, Daniel J; Kurlansky, Paul A; Burnett, John C

    2008-07-01

    Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides. Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP. Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP. Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation. The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides.

  16. Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

    Science.gov (United States)

    Vasconcelos, Andrea Rodrigues; Kinoshita, Paula Fernanda; Yshii, Lidia Mitiko; Marques Orellana, Ana Maria; Böhmer, Ana Elisa; de Sá Lima, Larissa; Alves, Rosana; Andreotti, Diana Zukas; Marcourakis, Tania; Scavone, Cristoforo; Kawamoto, Elisa Mitiko

    2015-05-01

    Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Mechanism and activation for allosteric adenosine 5'-monophosphate nucleosidase. Kinetic alpha-deuterium isotope effects for the enzyme-catalyzed hydrolysis of adenosine 5'-monophosphate and nicotinamide mononucleotide

    International Nuclear Information System (INIS)

    Skoog, M.T.

    1986-01-01

    The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents

  18. 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

    1977-01-01

    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. PMID:196595

  19. Use of nucleotides in weanling rats with diarrhea induced by a lactose overload: effect on the evolution of diarrhea and weight and on the histopathology of intestine, liver and spleen

    Directory of Open Access Journals (Sweden)

    Norton R.

    2001-01-01

    Full Text Available Until recently, dietary sources of nucleotides were thought not to be essential for good nutrition. Certain states with higher metabolic demands may require larger amounts that cannot be provided by endogenous production. The objective of the present study was to determine the action of nucleotides on the recovery from lactose-induced diarrhea in weaned rats. Thirty-six weanling Fisher rats were divided into two groups. Group 1 received a standard diet and group 2 received a diet containing lactose in place of starch. On the 10th day, six animals per group were sacrificed for histopathological evaluation. The remaining animals were divided into two other subgroups, each with 6 animals, receiving a control diet, a control diet with nucleotides (0.05% adenosine monophosphate, 0.05% guanosine monophosphate, 0.05% cytidine monophosphate, 0.05% uridine monophosphate and 0.05% inosine monophosphate, a diet with lactose, and a diet with lactose and nucleotides. On the 32nd day of the experiment all animals were sacrificed. Animals with diarrhea weighed less than animals without diarrhea. The introduction of nucleotides did not lead to weight gain. Mean diet consumption was lower in the group that continued to ingest lactose, with the group receiving lactose plus nucleotides showing a lower mean consumption. Animals receiving lactose had inflammatory reaction and deposits of periodic acid-Schiff-positive material in intestinal, hepatic and splenic tissues. The introduction of nucleotides led to an improvement of the intestinal inflammatory reaction. In lactose-induced diarrhea, when the stimulus is maintained - lactose overload - the nucleotides have a limited action on the weight gain and on recovery of intestinal morphology, although they have a protective effect on hepatic injury and improve the inflammatory response.

  20. Enhancement of Nucleoside Production in Hirsutella sinensis Based on Biosynthetic Pathway Analysis

    Science.gov (United States)

    Liu, Zhi-Qiang; Zhang, Bo; Lin, Shan; Baker, Peter James; Chen, Mao-Sheng; Xue, Ya-Ping; Wu, Hui; Xu, Feng; Yuan, Shui-Jin; Teng, Yi; Wu, Ling-Fang

    2017-01-01

    To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis. PMID:29333435

  1. 5'-Monophosphate-activated protein kinase (AMPK) improves autophagic activity in diabetes and diabetic complications.

    Science.gov (United States)

    Yao, Fan; Zhang, Ming; Chen, Li

    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.

  2. Selenomethionine substitution of orotidine-5-monophosphate decarboxylase causes a change in crystal contacts and space group

    DEFF Research Database (Denmark)

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

    2001-01-01

    with the inhibitor 1-(5'-phospho- -D-ribofuranosyl)barbituric acid crystallizes under similar conditions as the native enzyme. In contrast to the native enzyme, where the crystals belong to the orthorhombic space group P212121, the SeMet-substituted enzyme crystallizes in the monoclinic space group P21......-wavelength anomalous dispersion technique, both native and SeMet-substituted proteins have been produced and purified. During the production of SeMet ODCase, it was observed that SeMet was the only amino acid that it was necessary to add to the defined medium during expression. SeMet-substituted ODCase in complex...

  3. The combined effect of fludarabine monophosphate and radiation on lymphatic cell lines in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Nitsche, M.; Rave-Fraenk, M.; Wolter, C.; Hess, C.F. [Gottingen Univ. Georg-August, Dept. of Radiation Oncology (Germany); Pradier, O. [Centre Hospitalier Universitaire, Dept. de Cancerologie, 29 - Brest (France)

    2006-11-15

    We could demonstrate a fludarabine-induced radiosensitization in two of four cell lines tested. We conclude, that fludarabine may play a role as radiosensitizer. The combined effect of low dose fludarabine and low dose radiation emphasizes the idea of possible multiple reapplication of radio chemotherapy in progressive lymphoma. Further investigations are warranted to identify the potential of this drug as a radiosensitizer in vivo and to elucidate the mechanism of interaction of the drug and radiation. (N.C.)

  4. Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)].

    Science.gov (United States)

    Kaye, Karl; Bryant, David E; Marriott, Katie E R; Ohara, Shohei; Fishwick, Colin W G; Kee, Terence P

    2016-11-01

    We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H 2 P 2 O 5 2- ; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M 2+ ) and by organic co-factors such as acetate (AcO - ). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M 2+ & AcO - . Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

  5. Catalytic dephosphorylation of adenosine monophosphate (AMP) to form supramolecular nanofibers/hydrogels.

    Science.gov (United States)

    Du, Xuewen; Li, Junfeng; Gao, Yuan; Kuang, Yi; Xu, Bing

    2012-02-18

    The use of enzyme to instruct the self-assembly of the nucleoside of adenosine in water provides a new class of molecular nanofibers/hydrogels as functional soft materials. This journal is © The Royal Society of Chemistry 2012

  6. Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica.

    Science.gov (United States)

    Dieni, Christopher A; Storey, Kenneth B

    2008-04-22

    The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65-70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to approximately 1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg.ATP and Mg.ADP and inhibited by Mg.GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing. Maintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.

  7. Geminal hydroxy phosphonate derivatives of nucleosides: A novel class of nucleoside 5'-monophosphate analogues

    Czech Academy of Sciences Publication Activity Database

    Králíková, Šárka; Buděšínský, Miloš; Masojídková, Milena; Rosenberg, Ivan

    2000-01-01

    Roč. 41, č. 6 (2000), s. 955-958 ISSN 0040-4039 R&D Projects: GA MŠk LB98233 Grant - others:NIH(US) R03 TW00673-01 Institutional research plan: CEZ:AV0Z4055905 Subject RIV: CC - Organic Chemistry Impact factor: 2.558, year: 2000

  8. 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

    /ß-barrels with two shared active sites. The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out. The proximity of the OMP carboxylate group with Asp71 appears to be instrumental...

  9. Monophosphate tungsten bronzes with pentagonal tunnels: reinvestigation through the peephole of the superspace

    Czech Academy of Sciences Publication Activity Database

    Pérez, O.; Elcoro, L.; Pérez-Mato, J. M.; Petříček, Václav

    2013-01-01

    Roč. 69, č. 2 (2013), s. 122-136 ISSN 0108-7681 Grant - others:AV ČR(CZ) AP0701 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:68378271 Keywords : superspace theory * crystal structure analysis Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 2.095, year: 2013

  10. "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.

  11. Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

    Science.gov (United States)

    Tuder, R M; Karasek, M A; Bensch, K G

    1990-02-01

    The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

  12. Regulation of phospholipid synthesis in Mycobacterium smegmatis by cyclic adenosine monophosphate

    International Nuclear Information System (INIS)

    Sareen, Monica; Kaur, Harpinder; Khuller, G.K.

    1993-01-01

    Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis in Mycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [ 14 C]acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria. (author). 14 refs., 4 tabs

  13. Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, R.; Evans, G.; Rotella, F. J.; Westbrook, E. M.; Beno, D.; Huberman, E.; Joachimiak, A.; Collart, F. R.

    1999-01-01

    IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K{sub m} for NAD (1180 {mu}M) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 {angstrom} with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione {beta}-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

  14. Pharmacodynamic Monitoring of Inosine Monophosphate Dehydrogenase Activity: A Basis For Optimized and Individualized Mycophenolate Mofetil Therapy

    NARCIS (Netherlands)

    F. Sombogaard (Ferdi)

    2010-01-01

    textabstractIn 1896 Gosic isolated for the first time mycophenolic acid (MPA) from Penicillium glaucum.It was subsequently isolated from Penicillium stoloniferum Thom (synonym P. brevi-compactum Dierckx) by Alsberg and Black in 1913 who gave the acid phenolic substance its name mycophenolic acid

  15. 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

    . The yields of these were measured as a function of the wavelength of the light from 210 nm to 300 nm, and they were combined to obtain the total photoinduced dissociation at each wavelength (i.e., action spectrum). A broad band between 230 nm and 290 nm and the tail of a band with maximum below 210 nm (high......-energy band) are seen. In the case of AdeH+(H2O), the dominant dissociation channel after photoexcitation in the low-energy band was simply loss of H2O while photodissociation of protonated AMP revealed two dominant dissociation channels associated with the formation of either AdeH+ or loss of H3PO4....... The action spectra of AdeH+, AdeH+(H2O), and AMPH+ are almost identical in the 230–290 nm region, and they resemble the absorption spectrum of protonated adenine in aqueous solution recorded at low pH. Hence from our work it is firmly established that the lowest-energy transitions are independent...

  16. Resonant Infrared Multiple Photon Dissociation Spectroscopy of Anionic Nucleotide Monophosphate Clusters

    Czech Academy of Sciences Publication Activity Database

    Ligare, M. R.; Rijs, A. M.; Berden, G.; Kabeláč, M.; Nachtigallová, Dana; Oomens, J.; de Vries, M. S.

    2015-01-01

    Roč. 119, č. 25 (2015), s. 7894-7901 ISSN 1520-6106 R&D Projects: GA ČR GAP208/12/1318 Institutional support: RVO:61388963 Keywords : DNA polymerase beta * gas-phase conformations * excited-state dynamics Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.187, year: 2015

  17. Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

    Science.gov (United States)

    Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

    2016-05-03

    In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

  18. A selective phosphodiesterase 10A inhibitor reduces l-dopa-induced dyskinesias in parkinsonian monkeys.

    Science.gov (United States)

    Beck, Goichi; Maehara, Shunsuke; Chang, Phat Ly; Papa, Stella M

    2018-03-06

    Phosphodiesterase 10A is a member of the phosphodiesterase family whose brain expression is restricted to the striatum. Phosphodiesterase 10A regulates cyclic adenosine monophosphate and cyclic guanosine monophosphate, which mediate responses to dopamine receptor activation, and the levels of these cyclic nucleotides are decreased in experimental models of l-dopa-induced dyskinesia. The elevation of cyclic adenosine monophosphate/cyclic guanosine monophosphate levels by phosphodiesterase 10A inhibition may thus be targeted to reduce l-dopa-induced dyskinesia. The present study was aimed at determining the potential antidyskinetic effects of phosphodiesterase 10A inhibitors in a primate model of Parkinson's disease (PD). The experiments performed in this model were also intended to provide translational data for the design of future clinical trials. Five MPTP-treated macaques with advanced parkinsonism and reproducible l-dopa-induced dyskinesia were used. MR1916, a selective phosphodiesterase 10A inhibitor, at doses 0.0015 to 0.05 mg/kg, subcutaneously, or its vehicle (control test) was coadministered with l-dopa methyl ester acutely (predetermined optimal and suboptimal subcutaneous doses) and oral l-dopa chronically as daily treatment for 5 weeks. Standardized scales were used to assess motor disability and l-dopa-induced dyskinesia by blinded examiners. Pharmacokinetics was also examined. MR1916 consistently reduced l-dopa-induced dyskinesia in acute tests of l-dopa optimal and suboptimal doses. Significant effects were present with every MR1916 dose tested, but the most effective was 0.015 mg/kg. None of the MR1916 doses tested affected the antiparkinsonian action of l-dopa at the optimal dose. The anti-l-dopa-induced dyskinesia effect of MR1916 (0.015 mg/kg, subcutaneously) was sustained with chronic administration, indicating that tolerance did not develop over the 5-week treatment. No adverse effects were observed after MR1916 administration acutely or

  19. Cerebral haemodynamic response or excitability is not affected by sildenafil

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Hansen, Adam E; Larsson, Henrik B W

    2009-01-01

    Sildenafil (Viagra), a cyclic guanosine monophosphate-degrading phosphodiesterase 5 inhibitor, induces headache and migraine. Such headache induction may be caused by an increased neuronal excitability, as no concurrent effect on cerebral arteries is found. In 13 healthy females (23+/-3 years, 70......) were performed. The measurements were applied at baseline and at both 1 and 2 h after ingestion of 100 mg of sildenafil. Blood pressure, heart rate and side effects, including headache, were obtained. Headache was induced in all but one subject on both study days. Sildenafil did not affect VEP...... amplitude or latency (P100). The fMRI response to visual stimulation or hypercapnia was unchanged by sildenafil. In conclusion, sildenafil induces mild headache without potentiating a neuronal or local cerebrovascular visual response or a global cerebrovascular hypercapnic response. The implication...

  20. Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii

    International Nuclear Information System (INIS)

    Arnfors, Linda; Hansen, Thomas; Meining, Winfried; Schönheit, Peter; Ladenstein, Rudolf

    2005-01-01

    Nucleoside kinase from the hyperthermophilic archaeon M. jannaschii is a member of the PFK-B family which belongs to the ribokinase superfamily. Here, its expression, purification, crystallization and preliminary X-ray analysis are described. Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg 2+ . The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 Å for the apoenzyme crystals and 1.93 Å for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer

  1. (1)H, (13)C, (15)N backbone and side-chain resonance assignment of Nostoc sp. C139A variant of the heme-nitric oxide/oxygen binding (H-NOX) domain.

    Science.gov (United States)

    Alexandropoulos, Ioannis I; Argyriou, Aikaterini I; Marousis, Kostas D; Topouzis, Stavros; Papapetropoulos, Andreas; Spyroulias, Georgios A

    2016-10-01

    The H-NOX (Heme-nitric oxide/oxygen binding) domain is conserved across eukaryotes and bacteria. In human soluble guanylyl cyclase (sGC) the H-NOX domain functions as a sensor for the gaseous signaling agent nitric oxide (NO). sGC contains the heme-binding H-NOX domain at its N-terminus, which regulates the catalytic site contained within the C-terminal end of the enzyme catalyzing the conversion of GTP (guanosine 5'-triphosphate) to GMP (guanylyl monophosphate). Here, we present the backbone and side-chain assignments of the (1)H, (13)C and (15)N resonances of the 183-residue H-NOX domain from Nostoc sp. through solution NMR.

  2. The role of nitric oxide in reproduction

    Directory of Open Access Journals (Sweden)

    McCann S.M.

    1999-01-01

    Full Text Available Nitric oxide (NO plays a crucial role in reproduction at every level in the organism. In the brain, it activates the release of luteinizing hormone-releasing hormone (LHRH. The axons of the LHRH neurons project to the mating centers in the brain stem and by afferent pathways evoke the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine monophosphate (cGMP. NO also activates the release of LHRH which reaches the pituitary and activates the release of gonadotropins by activating neural NO synthase (nNOS in the pituitary gland. In the gonad, NO plays an important role in inducing ovulation and in causing luteolysis, whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it via prostaglandins (PG.

  3. Perturbing Tandem Energy Transfer in Luminescent Heterobinuclear Lanthanide Coordination Polymer Nanoparticles Enables Real-Time Monitoring of Release of the Anthrax Biomarker from Bacterial Spores.

    Science.gov (United States)

    Gao, Nan; Zhang, Yunfang; Huang, Pengcheng; Xiang, Zhehao; Wu, Fang-Ying; Mao, Lanqun

    2018-06-05

    Lanthanide-based luminescent sensors have been widely used for the detection of the anthrax biomarker dipicolinic acid (DPA). However, mainly based on DPA sensitization to the lanthanide core, most of them failed to realize robust detection of DPA in bacterial spores. We proposed a new strategy for reliable detection of DPA by perturbing a tandem energy transfer in heterobinuclear lanthanide coordination polymer nanoparticles simply constructed by two kinds of lanthanide ions, Tb 3+ and Eu 3+ , and guanosine 5'-monophosphate. This smart luminescent probe was demonstrated to exhibit highly sensitive and selective visual luminescence color change upon exposure to DPA, enabling accurate detection of DPA in complex biosystems such as bacterial spores. DPA release from bacterial spores on physiological germination was also successfully monitored in real time by confocal imaging. This probe is thus expected to be a powerful tool for efficient detection of bacterial spores in responding to anthrax threats.

  4. Effect of sildenafil on gastric emptying and postprandial frequency of antral contractions in healthy humans

    DEFF Research Database (Denmark)

    Madsen, J L; Søndergaard, S B; Fuglsang, S

    2004-01-01

    BACKGROUND: Sildenafil is known to block phosphodiesterase type 5, which degrades nitric oxide-stimulated cyclic guanosine monophosphate, thereby relaxing smooth muscle cells in various organs. The effect of sildenafil on gastric motor function after a meal was investigated in healthy humans....... METHODS: Ten healthy male volunteers (21-28 years) participated in a placebo-controlled, double-blind, cross-over study. In random order and on two separate days each volunteer ingested either 50 mg sildenafil (Viagra, Pfizer, New York, N.Y., USA) or placebo. A gamma camera technique was used to measure......: A single dose of 50 mg sildenafil does not change gastric emptying or postprandial frequency of antral contractions in healthy volunteers....

  5. Crystallization and preliminary X-ray analysis of the flagellar motor `brake' molecule YcgR with c-di-GMP from Escherichia coli.

    Science.gov (United States)

    Hou, Yanjie; Li, De Feng; Wang, Da Cheng

    2013-06-01

    In Escherichia coli and Salmonella enterica, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous bacterial second-messenger molecule that participates in many cellular processes, can regulate flagellar motor speed and reduce cell swimming velocity by binding to the PilZ-containing protein YcgR. Here, the crystallization and preliminary X-ray crystallographic analysis of YcgR with c-di-GMP are reported. The crystals diffracted to 2.3 Å resolution and belonged to space group R3:H, with unit-cell parameters a = b = 93.96, c = 109.61 Å. The asymmetric unit appeared to contain one subunit with a Matthews coefficient of 3.21 Å(3) Da(-1). The results reported here provide a sound basis for solving the crystal structure of YcgR with c-di-GMP and revealing its structure-function relationship based on the three-dimensional structure.

  6. Potentiation of cGMP signaling increases oxygen delivery and oxidative metabolism in contracting skeletal muscle of older but not young humans

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Piil, Peter Bergmann; Egelund, Jon

    2015-01-01

    regulation remain unresolved. Cyclic guanosine monophosphate (cGMP) is one of the main second messengers that mediate smooth muscle vasodilation and alterations in cGMP signaling could, therefore, be one mechanism by which skeletal muscle perfusion is impaired with advancing age. The current study aimed...... to evaluate the effect of inhibiting the main enzyme involved in cGMP degradation, phosphodiesterase 5 (PDE5), on blood flow and O2 delivery in contracting skeletal muscle of young and older humans. A group of young (23 ± 1 years) and a group of older (72 ± 2 years) male human subjects performed submaximal...... in the older subjects correlated with the increase in leg O2 uptake (r (2) = 0.843). These findings suggest an insufficient O2 delivery to the contracting skeletal muscle of aged individuals and that reduced cGMP availability is a novel mechanism underlying impaired skeletal muscle perfusion with advancing age....

  7. Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

    DEFF Research Database (Denmark)

    Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

    2007-01-01

    waves sweeping through the cytoplasm when the SR is stimulated to release calcium. A rise in cyclic guanosine monophosphate (cGMP) leads to the experimentally observed transition from waves to whole-cell calcium oscillations. At the same time membrane potential starts to oscillate and the frequency...... approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion. Key words: Vasomotion, Chloride channel, cGMP, Mathematical model, Calcium waves....

  8. The role of cGMP hydrolysing phosphodiesterases 1 and 5 in cerebral artery dilatation

    DEFF Research Database (Denmark)

    Kruuse, Christina; Rybalkin, S D; Khurana, T S

    2001-01-01

    The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (cGMP...... a close relation to the nitric oxide-cGMP pathway. The responses to zaprinast and dipyridamole, however, were not only moderately affected, but also restored by sodium nitroprusside (0.1 microM) pretreatment. At high concentrations, the dilatory effects of zaprinast and dipyridamole were partly caused...... by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease....

  9. Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

    Science.gov (United States)

    Matsushika, Akinori; Nagashima, Atsushi; Goshima, Tetsuya; Hoshino, Tamotsu

    2013-01-01

    In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4

  10. Association of fat mass and obesity-associated and retinitis pigmentosa guanosine triphosphatase (GTPase) regulator-interacting protein-1 like polymorphisms with body mass index in Chinese women.

    Science.gov (United States)

    Chen, Boyu; Li, Zhiqiang; Chen, Jianhua; Ji, Jue; Shen, Jingyi; Xu, Yufeng; Zhao, Yingying; Liu, Danping; Shen, Yinhuan; Zhang, Weijie; Shen, Jiawei; Wang, Yonggang; Shi, Yongyong

    2018-04-14

    Body mass index (BMI) is the most commonly used quantitative measure of adiposity. It is a kind of complex genetic diseases which are caused by multiple susceptibility genes. The first intron of fat mass and obesity-associated (FTO) has been widely discovered to be associated with BMI. Retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L) is located in the upstream region of FTO and has been proved to be linked with obesity through functional tests. We carried out a genetic association analysis to figure out the role of the FTO gene and the RPGRIP1L gene in BMI. A quantitative traits study with 6,102 Chinese female samples, adjusted for age, was performed during our project. Among the twelve SNPs, rs1421085, rs1558902, rs17817449, rs8050136, rs9939609, rs7202296, rs56137030, rs9930506 and rs12149832 in the FTO gene were significantly associated with BMI after Bonferroni correction. Meanwhile, rs9934800 in the RPGRIP1L gene showed significance with BMI before Bonferroni correction, but this association was eliminated after Bonferroni correction. Our results suggested that genetic variants in the FTO gene were strongly associated with BMI in Chinese women, which may serve as targets of pharmaceutical research and development concerning BMI. Meanwhile, we didn't found the significant association between RPGRIP1L and BMI in Chinese women.

  11. Estimating the Distribution and Production of Microplankton in a Coastal Upwelling Front from the Cellular Content of Guanosine-5’-Triphosphate and Adenosine-5’-Triphosphate.

    Science.gov (United States)

    1981-09-01

    through a microfine glass fiber tilter (Reeve Angel 984-H with a pore size of approximately 0.45 pnm). This...buffer (pH 7.7) in order to extract the nucleotides present in the water sample. The test tubes containing the extracts were labeled and stored frozen...into a series of disposable glass cuvettes (12x75mm) and placed in a test tube rack in an incubating water bath at 300 C. Each tube was allowed to

  12. Automated method for the direct analysis of 8-oxo-guanosine and 8-oxo-2'-deoxyguanosine in human urine using ultraperformance liquid chromatography and tandem mass spectrometry

    DEFF Research Database (Denmark)

    Henriksen, Trine; Hillestrøm, Peter R; Poulsen, Henrik E

    2009-01-01

    The potential use of oxidative stress-induced DNA and RNA damage products as biomarkers is an important aspect of biomedical research. There is a need for assays with high specificity and sensitivity that also can be used in molecular epidemiology studies with a large number of subjects. In addit......The potential use of oxidative stress-induced DNA and RNA damage products as biomarkers is an important aspect of biomedical research. There is a need for assays with high specificity and sensitivity that also can be used in molecular epidemiology studies with a large number of subjects...

  13. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

    International Nuclear Information System (INIS)

    Baldassare, J.J.; Henderson, P.A.; Fisher, G.J.

    1989-01-01

    The effects of thrombin and GTPγS on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [ 3 H]inositol-labeled membranes or with lipid vesicles containing either [ 3 H]phosphatidylinositol or [ 3 H]phosphatidylinositol 4,5-bisphosphate. GTPγS (1 μM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP 3 ), inositol bisphosphate (IP 2 ), or inositol phosphate (IP) from [ 3 H]inositol-labeled membranes. IP 2 and IP 3 , but not IP, from [ 3 H]inositol-labeled membranes were, however, stimulated 3-fold by GTPγS (1 μM) plus thrombin (1 unit/mL). A higher concentration of GTPγS (100 μM) alone also stimulated IP 2 and IP 3 , but not IP, release. In the presence of 1 mM calcium, release of IP 2 and IP 3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by platelet membrane associated PLC was also markedly enhanced by GTPγS (100 μM) or GTPγS (1 μM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP 2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTPγS (100 μM) or calcium (1 mM) dependent PIP 2 breakdown, while TPA inhibited GTPγS-dependent but not calcium-dependent phospholipase C activity

  14. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    International Nuclear Information System (INIS)

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-01-01

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [ 32 P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-α/sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-α/sub i/ or anti-α 0 antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed

  15. DNA sensor cGAS-mediated immune recognition

    Directory of Open Access Journals (Sweden)

    Pengyan Xia

    2016-09-01

    Full Text Available Abstract The host takes use of pattern recognition receptors (PRRs to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.

  16. Role of fetal DNA in preeclampsia (review).

    Science.gov (United States)

    Konečná, Barbora; Vlková, Barbora; Celec, Peter

    2015-02-01

    Preeclampsia is an autoimmune disorder characterized by hypertension. It begins with abnormal cytotrophoblast apoptosis, which leads to inflammation and an increase in the levels of anti-angiogenic factors followed by the disruption of the angiogenic status. Increased levels of fetal DNA and RNA coming from the placenta, one of the most commonly affected organs in pregnancies complicated by preeclampsia, have been found in pregnant women with the condition. However, it remains unknown as to whether this is a cause or a consequence of preeclampsia. Few studies have been carried out on preeclampsia in which an animal model of preeclampsia was induced by an injection of different types of DNA that are mimic fetal DNA and provoke inflammation through Toll-like receptor 9 (TLR9) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The specific mechanisms involved in the development of preeclampsia are not yet fully understood. It is hypothesized that the presence of different fragments of fetal DNA in maternal plasma may cause for the development of preeclampsia. The function of DNase during preeclampsia also remains unresolved. Studies have suggested that its activity is decreased or the DNA is protected against its effects. Further research is required to uncover the pathogenesis of preeclampsia and focus more on the condition of patients with the condition.

  17. Increasing plasma fibrinogen, but unchanged levels of intraplatelet cyclic nucleotides, plasma endothelin-1, factor VII, and neopterin during cholesterol lowering with fluvastatin.

    Science.gov (United States)

    Gottsäter, A; Anwaar, I; Lind, P; Mattiasson, I; Lindgärde, F

    1999-04-01

    Lipid-lowering statin treatment reduces cardiovascular morbidity and mortality and improves endothelial function in patients with hypercholesterolemia. The aim of the present study was to evaluate plasma levels of fibrinogen, factor VII, and the macrophage-derived inflammatory mediator neopterin during lipid lowering. In addition, the endothelial production of platelet antiaggregatory and vasodilatory factors such as nitric oxide and prostacyclin, and vasoconstrictive factors such as endothelin-1, was assessed. Plasma fibrinogen, factor VII, endothelin-1, and the neopterin and intraplatelet nitric oxide and prostacyclin mediators cyclic 3'-5'guanosine monophosphate (cGMP) and cyclic 3'-5'adenosine monophosphate (cAMP) were measured before and 6 months after the institution of treatment with fluvastatin in 17 patients (eight men and nine women, median age 60 years) with vascular disease and previously untreated hypercholesterolemia. After 6 months, a decrease of 1.62 mmol/l [1.26-2.18 (19%); P factor VII [from 1.14 IE/ml (0.58-1.38) to 1.22 IE/ml (0.96-1.46); NS], or plasma neopterin [from 8.6 nmol/l (7.1-11.5) to 8.7 nmol/l (7.9-11.3); NS]. In conclusion, during cholesterol-lowering treatment with fluvastatin, plasma levels of fibrinogen increased whereas intraplatelet cyclic nucleotide levels and plasma endothelin-1, factor VII and neopterin levels were unchanged.

  18. Formulation of yeast-leavened bread with reduced salt content by using a Lactobacillus plantarum fermentation product.

    Science.gov (United States)

    Valerio, Francesca; Conte, Amalia; Di Biase, Mariaelena; Lattanzio, Veronica M T; Lonigro, S Lisa; Padalino, Lucia; Pontonio, Erica; Lavermicocca, Paola

    2017-04-15

    A Lactobacillus plantarum fermentation product (Bio21B), obtained after strain growth (14h) in a wheat flour-based medium, was applied in the bread-making process as taste enhancer, in order to obtain a yeast-leavened bread with reduced salt content (20% and 50%) with respect to a reference bread (REF) not containing the fermentation product. Sensory analysis indicated that the Bio21B bread with salt reduced by 50% had a pleasant taste similar to the salt-containing bread (REF). l-Glutamate and total free amino acid content did not differ between REF and Bio21B breads, while the acids lactic, acetic, phenyllactic, 4-OH-phenyllactic and indole-3-lactic were present only in Bio21B breads. Moreover, the presence of several umami (uridine monophosphate, inosine monophosphate, adenosine, and guanosine) and kokumi (γ-l-glutamyl-l-valine) taste-related molecules was ascertained both in REF and in Bio21B breads. Therefore, a possible role of the acidic molecules in compensating the negative perception of salt reduction can be hypothesized. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Ex vivo relaxation effect of Cuscuta chinensis extract on rabbit corpus cavernosum.

    Science.gov (United States)

    Sun, Kai; Zhao, Chen; Chen, Xiang-Feng; Kim, Hye-Kyung; Choi, Bo-Ram; Huang, Yi-Ran; Park, Jong-Kwan

    2013-01-01

    The effect of Cuscuta chinensis extract on the rabbit penile corpus cavernosum (PCC) was evaluated in the present study. Penises obtained from healthy male New Zealand white rabbits (2.5-3.0 kg) were precontracted with phenylephrine (Phe, 10 µmol l(-1)) and then treated with various concentrations of Cuscuta chinensis extract (1, 2, 3, 4 and 5 mg ml(-1)). The change in penile tension was recorded, and cyclic nucleotides in the PCC were measured by radioimmunoassay (RIA). The interaction between Cuscuta chinensis and sildenafil was also evaluated. The result indicated that the PCC relaxation induced by Cuscuta chinensis extract was concentration-dependent. Pre-treatment with an nitric oxide synthase (NOS) inhibitor (Nω nitro-L-arginine-methyl ester, L-NAME), a guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ), or a protein kinase A inhibitor (KT 5720) did not completely inhibit the relaxation. Incubation of penile cavernous tissue with the Cuscuta chinensis extract significantly increased cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in the PCC. Moreover, the Cuscuta chinensis extract significantly enhanced sildenafil-induced PCC relaxation. In conclusion, the Cuscuta chinensis extract exerts a relaxing effect on penile cavernous tissue in part by activating the NO-cGMP pathway, and it may improve erectile dysfunction (ED), which does not completely respond to sildenafil citrate.

  20. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Joong-Won, E-mail: jshin@govst.edu [Division of Science, Governors State University, University Park, Illinois 60484-0975 (United States); Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States); Bernstein, Elliot R., E-mail: erb@lamar.colostate.edu [Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States)

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  1. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    International Nuclear Information System (INIS)

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-01

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5 ′ -monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results

  2. Transmission of innate immune signaling by packaging of cGAMP in viral particles.

    Science.gov (United States)

    Gentili, Matteo; Kowal, Joanna; Tkach, Mercedes; Satoh, Takeshi; Lahaye, Xavier; Conrad, Cécile; Boyron, Marilyn; Lombard, Bérangère; Durand, Sylvère; Kroemer, Guido; Loew, Damarys; Dalod, Marc; Théry, Clotilde; Manel, Nicolas

    2015-09-11

    Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells. Copyright © 2015, American Association for the Advancement of Science.

  3. An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway.

    Science.gov (United States)

    Bose, Debojit; Su, Yichi; Marcus, Assaf; Raulet, David H; Hammond, Ming C

    2016-12-22

    In mammalian cells, the second messenger (2'-5',3'-5') cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP), is produced by the cytosolic DNA sensor cGAMP synthase (cGAS), and subsequently bound by the stimulator of interferon genes (STING) to trigger interferon response. Thus, the cGAS-cGAMP-STING pathway plays a critical role in pathogen detection, as well as pathophysiological conditions including cancer and autoimmune disorders. However, studying and targeting this immune signaling pathway has been challenging due to the absence of tools for high-throughput analysis. We have engineered an RNA-based fluorescent biosensor that responds to 2',3'-cGAMP. The resulting "mix-and-go" cGAS activity assay shows excellent statistical reliability as a high-throughput screening (HTS) assay and distinguishes between direct and indirect cGAS inhibitors. Furthermore, the biosensor enables quantitation of 2',3'-cGAMP in mammalian cell lysates. We envision this biosensor-based assay as a resource to study the cGAS-cGAMP-STING pathway in the context of infectious diseases, cancer immunotherapy, and autoimmune diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

    Science.gov (United States)

    Kato, Kazuki; Omura, Hiroki; Ishitani, Ryuichiro; Nureki, Osamu

    2017-06-20

    The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

  5. Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

    Science.gov (United States)

    Collins, Angela C; Cai, Haocheng; Li, Tuo; Franco, Luis H; Li, Xiao-Dong; Nair, Vidhya R; Scharn, Caitlyn R; Stamm, Chelsea E; Levine, Beth; Chen, Zhijian J; Shiloh, Michael U

    2015-06-10

    Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The cGAS-cGAMP-STING pathway connects DNA damage to inflammation, senescence, and cancer.

    Science.gov (United States)

    Li, Tuo; Chen, Zhijian J

    2018-05-07

    Detection of microbial DNA is an evolutionarily conserved mechanism that alerts the host immune system to mount a defense response to microbial infections. However, this detection mechanism also poses a challenge to the host as to how to distinguish foreign DNA from abundant self-DNA. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates the adaptor protein STING. However, cGAS can be activated by double-stranded DNA irrespective of the sequence, including self-DNA. Although how cGAS is normally kept inactive in cells is still not well understood, recent research has provided strong evidence that genomic DNA damage leads to cGAS activation to stimulate inflammatory responses. This review summarizes recent findings on how genomic instability and DNA damage trigger cGAS activation and how cGAS serves as a link from DNA damage to inflammation, cellular senescence, and cancer. © 2018 Li and Chen.

  7. Apoptosis in mammalian oocytes: a review.

    Science.gov (United States)

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  8. Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone.

    Science.gov (United States)

    Detremmerie, Charlotte; Vanhoutte, Paul M; Leung, Susan

    2017-07-01

    The natural compound thymoquinone, extracted from Nigella sativa (black cumin), is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC) producing inosine 3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine 3',5'-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

  9. Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone

    Directory of Open Access Journals (Sweden)

    Charlotte Detremmerie

    2017-07-01

    Full Text Available The natural compound thymoquinone, extracted from Nigella sativa (black cumin, is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC producing inosine 3ʹ,5ʹ-cyclic monophosphate (cyclic IMP rather than guanosine 3ʹ,5ʹ-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

  10. Homology modeling, docking studies and molecular dynamic simulations using graphical processing unit architecture to probe the type-11 phosphodiesterase catalytic site: a computational approach for the rational design of selective inhibitors.

    Science.gov (United States)

    Cichero, Elena; D'Ursi, Pasqualina; Moscatelli, Marco; Bruno, Olga; Orro, Alessandro; Rotolo, Chiara; Milanesi, Luciano; Fossa, Paola

    2013-12-01

    Phosphodiesterase 11 (PDE11) is the latest isoform of the PDEs family to be identified, acting on both cyclic adenosine monophosphate and cyclic guanosine monophosphate. The initial reports of PDE11 found evidence for PDE11 expression in skeletal muscle, prostate, testis, and salivary glands; however, the tissue distribution of PDE11 still remains a topic of active study and some controversy. Given the sequence similarity between PDE11 and PDE5, several PDE5 inhibitors have been shown to cross-react with PDE11. Accordingly, many non-selective inhibitors, such as IBMX, zaprinast, sildenafil, and dipyridamole, have been documented to inhibit PDE11. Only recently, a series of dihydrothieno[3,2-d]pyrimidin-4(3H)-one derivatives proved to be selective toward the PDE11 isoform. In the absence of experimental data about PDE11 X-ray structures, we found interesting to gain a better understanding of the enzyme-inhibitor interactions using in silico simulations. In this work, we describe a computational approach based on homology modeling, docking, and molecular dynamics simulation to derive a predictive 3D model of PDE11. Using a Graphical Processing Unit architecture, it is possible to perform long simulations, find stable interactions involved in the complex, and finally to suggest guideline for the identification and synthesis of potent and selective inhibitors. © 2013 John Wiley & Sons A/S.

  11. Interaction of organophosphorus pesticides with DNA nucleotides on a Boron-doped diamond electrode

    Energy Technology Data Exchange (ETDEWEB)

    Garbellini, Gustavo S.; Uliana, Carolina V.; Yamanaka, Hideko, E-mail: gustgarb@yahoo.com.br [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Bauru, SP (Brazil). Dept. de Quimica Analitica

    2013-12-01

    Diamond electrode was used to evaluate the interaction of the nucleotides guanosine monophosphate (GMP) and adenosine monophosphate (AMP) with the pesticides chlorpyrifos, methamidophos and monocrotophos. Changes were observed in the currents and peak potentials of the nucleotide voltammograms in the presence of the pesticides, with dependence on the pesticide concentration (from 5.0 Multiplication-Sign 10{sup -7} to 5.0 Multiplication-Sign 10{sup -5} mol L{sup -1}) and the interaction time (from 1 min to 4 h). This is probably due to binding of the pesticides to the nitrogenous bases present in the nucleotides, which could lead to problems in the DNA replication and biological functions of nucleotides. The pesticides showed stronger interaction with AMP than with GMP. Studies of the interaction of 50 Micro-Sign g mL{sup -1} DNA with the pesticides (from 30 min to 4 h and from 1.0 Multiplication-Sign 10{sup -6} to 6.0 Multiplication-Sign 10{sup -5} mol L{sup -1}) did not reveal any peaks relating to double helix opening or DNA unwinding. (author)

  12. Phosphodiesterases regulate airway smooth muscle function in health and disease.

    Science.gov (United States)

    Krymskaya, Vera P; Panettieri, Reynold A

    2007-01-01

    On the basis of structure, regulation, and kinetic properties, phosphodiesterases (PDEs) represent a superfamily of enzymes divided into 11 subfamilies that catalyze cytosolic levels of 3',5'-cyclic adenosine monophosphate (cAMP) or 3',5'-cyclic guanosine monophosphate (cGMP) to 5'-AMP or 5'-GMP, respectively. PDE4 represents the major PDE expressed in inflammatory cells as well as airway smooth muscle (ASM), and selective PDE4 inhibitors provide a broad spectrum of anti-inflammatory effects such as abrogating cytokine and chemokine release from inflammatory cells and inhibiting inflammatory cell trafficking. Due to cell- and tissue-specific gene expression and regulation, PDEs modulate unique organ-based functions. New tools or compounds that selectively inhibit PDE subfamilies and genetically engineered mice deficient in selective isoforms have greatly enhanced our understanding of PDE function in airway inflammation and resident cell function. This chapter will focus on recent advances in our understanding of the role of PDE in regulating ASM function.

  13. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi [Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo 184-8588 (Japan); Fujii, Kentaro; Yokoya, Akinari [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki 319-1195 (Japan); Fukuda, Yoshihiro; Saitoh, Yuji [Synchrotron Radiation Research Center, Japan Atomic Energy Agency, Sayo-gun, Hyougo 679-5148 (Japan)

    2014-08-07

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5{sup ′}-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5{sup ′}-monophosphate, and adenosine 5{sup ′}-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  14. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  15. Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium.

    Science.gov (United States)

    Schlageter, N; Janis, R A; Gualtieri, R T; Hechter, O

    1980-03-01

    The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.

  16. Synthesis and degradation of cyclic nucleotides in brain after a high dose of ionizing radiation

    International Nuclear Information System (INIS)

    Hunt, W.A.; Dalton, T.K.

    1981-01-01

    Previous data from our laboratory have indicated that a high dose of ionizing radiation can deplete the cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) on several areas of the rat brain. cGMP is more sensitive to radiation than cAMP and does not recover for at least 24 h after irradiation. The response of cAMP is transient and recovery occurs within 4 h. The purpose of the present paper is to determine whether alternations in the activity of the synthetic and degradative enzymes that regulate cyclic nucleotide levels could account for the observed effects. Guanylate and adenylate cyclase and cGMP and cAMP phosphodiesterase activities were determined 10 min after irradiation with 10,000 rad of high-energy electrons. No alteration was detected under these experimental conditions. The data suggest that the reduction in cyclic nucleotides is not a direct effect on their metabolic enzymes and is probably secondary to some as yet-undefined action of radiation on the brain

  17. Pharmacotherapy of erectile dysfunction: Current standards

    Directory of Open Access Journals (Sweden)

    Kew-Kim Chew

    2006-01-01

    Full Text Available Pharmacotherapy is currently the therapeutic option of choice for erectile dysfunction. Comprising mainly intracavernosal injection therapy using alprostadil or alprostadil combined with phentolamine and/or papaverine and oral phosphodiesterase-5 inhibitors, it is safe and effective if appropriately prescribed and administered. The medications in current use produce satisfactory erectile responses by enhancing cavernosal vasodilatation mainly through their ability to promote relaxation of the smooth muscle cells in the corpora cavernosa involving the synthesis and activity of nitric oxide via the cyclic guanosine monophosphate and cyclic adenosine monophosphate biochemical pathways. The main side-effects and complications of intracavernosal injections are postinjection pain, prolonged erections, priapism and penile fibrosis. There may be a variety of side-effects with phosphodiesterase-5 inhibition but these are usually inconsequential. Recent serious ill health and the need for ongoing long-acting nitrate therapy or frequent use of short-acting nitrates for angina are absolute contraindications to the use of phosphodiesterase-5 inhibitors. Caution has to be exercised in prescribing phosphodiesterase-5 inhibitors for patients with impaired renal or hepatic functions or receiving multi-drug therapy for any systemic disease. All patients presenting with erectile dysfunction should be investigated and treated for cardiovascular risk factors. They should also be counseled regarding lifestyle factors particularly healthy balanced diet, regular physical exercise and inappropriate social habits.

  18. Vascular nitric oxide: Beyond eNOS

    Directory of Open Access Journals (Sweden)

    Yingzi Zhao

    2015-10-01

    Full Text Available As the first discovered gaseous signaling molecule, nitric oxide (NO affects a number of cellular processes, including those involving vascular cells. This brief review summarizes the contribution of NO to the regulation of vascular tone and its sources in the blood vessel wall. NO regulates the degree of contraction of vascular smooth muscle cells mainly by stimulating soluble guanylyl cyclase (sGC to produce cyclic guanosine monophosphate (cGMP, although cGMP-independent signaling [S-nitrosylation of target proteins, activation of sarco/endoplasmic reticulum calcium ATPase (SERCA or production of cyclic inosine monophosphate (cIMP] also can be involved. In the blood vessel wall, NO is produced mainly from l-arginine by the enzyme endothelial nitric oxide synthase (eNOS but it can also be released non-enzymatically from S-nitrosothiols or from nitrate/nitrite. Dysfunction in the production and/or the bioavailability of NO characterizes endothelial dysfunction, which is associated with cardiovascular diseases such as hypertension and atherosclerosis.

  19. Nitric oxide in the rat cerebellum after hypoxia/ischemia.

    Science.gov (United States)

    Rodrigo, José; Fernández, Ana Patricia; Alonso, David; Serrano, Julia; Fernández-Vizarra, Paula; Martínez-Murillo, Ricardo; Bentura, María Luisa; Martinez, Alfredo

    2004-01-01

    Nitric oxide is a regulatory biological substance and an important intracellular messenger that acts as a specific mediator of various neuropathological disorders. In mammals and invertebrates, nitric oxide is synthesized from L-arginine in the central and peripheral neural structures by the endothelial, neuronal and inducible enzymatic isoforms of nitric oxide synthase. Nitric oxide may affect the function of various neurotransmitter-specific systems, and is involved in neuromodulation, reproductive function, immune response, and regulation of the cerebral blood circulation. This makes nitric oxide the main candidate in brain responses to brain ischemia/hypoxia. The cerebellum has been reported to be the area of the brain that has the highest nitric oxide synthase activity and the highest concentration of glutamate and aspartate. By glutamate receptors and physiological action of nitric oxide, cyclic guanisine-5'-monophosphate may be rapidly increased. The cerebellum significantly differs with respect to ischemia and hypoxia, this response being directly related to the duration and intensity of the injury. The cerebellum could cover the eventual need for nitric oxide during the hypoxia, boosting the nitric oxide synthase activity, but overall ischemia would require de novo protein synthesis, activating the inducible nitric oxide synthase to cope with the new situation. The specific inhibitors of nitric oxide synthesis show neuroprotective effects.

  20. Improved circadian sleep-wake cycle in infants fed a day/night dissociated formula milk.

    Science.gov (United States)

    Cubero, J; Narciso, D; Aparicio, S; Garau, C; Valero, V; Rivero, M; Esteban, S; Rial, R; Rodríguez, A B; Barriga, C

    2006-06-01

    On the basis of the circadian nutritional variations present in breast milk, and of the implications for the sleep/wake cycle of the nutrients present in infant formula milks, we designed a formula milk nutritionally dissociated into a Day/Night composition. The goal was to improve the bottle-fed infant's sleep/wake circadian rhythm. A total of 21 infants aged 4-20 weeks with sleeping difficulties were enrolled in the three-week duration study. The sleep analysis was performed using an actimeter (Actiwatch) placed on an ankle of each infant to uninterruptedly record movements during the three weeks. The dissociated Day milk, designed to be administered from 06:00 to 18:00, contained low levels of tryptophan (1.5g/100g protein) and carbohydrates, high levels of proteins, and the nucleotides Cytidine 5 monophosphate, Guanosine 5 monophosphate and Inosine 5 monophosphate. The dissociated Night milk, designed to be administered from 18.00 to 06.00, contained high levels of tryptophan (3.4g/100g protein) and carbohydrates, low levels of protein, and the nucleotides Adenosine 5 monophosphate and Uridine 5 monophosphate. Three different milk-feeding experiments were performed in a double-blind procedure covering three weeks. In week 1 (control), the infants received both by day and by night a standard formula milk; in week 2 (inverse control), they received the dissociated milk inversely (Night/Day instead of Day/Night); and in week 3, they received the Day/Night dissociated formula concordant with the formula design. When the infants were receiving the Day/Night dissociated milk in concordance with their environment, they showed improvement in all the nocturnal sleep parameters analyzed: total hours of sleep, sleep efficiency, minutes of nocturnal immobility, nocturnal awakenings, and sleep latency. In conclusion, the use of a chronobiologically adjusted infant formula milk seems to be effective in improving the consolidation of the circadian sleep/wake cycle in bottle

  1. Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

    Science.gov (United States)

    Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

    1996-06-01

    We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

  2. Identification of key pathways and genes influencing prognosis in bladder urothelial carcinoma

    Directory of Open Access Journals (Sweden)

    Ning X

    2017-03-01

    Full Text Available Xin Ning, Yaoliang Deng Department of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Province, People’s Republic of China Background: Genomic profiling can be used to identify the predictive effect of genomic subsets for determining prognosis in bladder urothelial carcinoma (BUC after radical cystectomy. This study aimed to investigate potential gene and pathway markers associated with prognosis in BUC.Methods: A microarray dataset of BUC was obtained from The Cancer Genome Atlas database. Differentially expressed genes (DEGs were identified by DESeq of the R platform. Kaplan–Meier analysis was applied for prognostic markers. Key pathways and genes were identified using bioinformatics tools, such as gene set enrichment analysis, gene ontology, the Kyoto Encyclopedia of Genes and Genomes, gene multiple association network integration algorithm (GeneMANIA, Search Tool for the Retrieval of Interacting Genes/Proteins, and Molecular Complex Detection.Results: A comparative gene set enrichment analysis of tumor and adjacent normal tissues suggested BUC tumorigenesis resulted mainly from enrichment of cell cycle and DNA damage and repair-related biological processes and pathways, including TP53 and mitotic recombination. Two hundred and fifty-six genes were identified as potential prognosis-related DEGs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the potential prognosis-related DEGs were enriched in angiogenesis, including the cyclic adenosine monophosphate biosynthetic process, cyclic guanosine monophosphate-protein kinase G, mitogen-activated protein kinase, Rap1, and phosphoinositide-3-kinase-AKT signaling pathway. Nine hub genes, TAGLN, ACTA2, MYH11, CALD1, MYLK, GEM, PRELP, TPM2, and OGN, were identified from the intersection of protein–protein interaction and GeneMANIA networks. Module analysis of protein–protein interaction and GeneMANIA networks mainly showed

  3. Presence of cyclic adenosine-3':5'-monophosphate (cAMP) in primary shoots of Zea mays L

    International Nuclear Information System (INIS)

    Edlich, W.; Graeser, H.

    1978-01-01

    The concentration of cAMP was determined in extracts of crude homogenates of maize seedlings, 3800 . g supernatants and isolated chloroplasts by the isotope dilution test. After extractpurification by precipitation with BaSO 4 and by chromatography on aluminium oxide column, a factor by means of which the binding of [8- 3 H]-cAMP with cAMP-dependent protein kinase was inhibited. The inhibitor was found inactive after treatment of the extracts with phosphodiesterase. In conclusion, this factor was identical with cAMP. It is suggested that cAMP-synthesis is localized in chloroplasts. Microbial contaminations which might disturb the detection of cAMP was excluded at least in the preparations of chloroplasts. (author)

  4. Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A : a prospective cohort study

    NARCIS (Netherlands)

    Schwahn, Bernd C.; Van Spronsen, Francjan J.; Belaidi, Abdel A.; Bowhay, Stephen; Christodoulou, John; Derks, Terry G.; Hennermann, Julia B.; Jameson, Elisabeth; Koenig, Kai; McGregor, Tracy L.; Font-Montgomery, Esperanza; Santamaria-Araujo, Jose A.; Santra, Saikat; Vaidya, Mamta; Vierzig, Anne; Wassmer, Evangeline; Weis, Ilona; Wong, Flora Y.; Veldman, Alex; Schwarz, Guenter

    2015-01-01

    Background Molybdenum cofactor deficiency (MoCD) is characterised by early, rapidly progressive postnatal encephalopathy and intractable seizures, leading to severe disability and early death. Previous treatment attempts have been unsuccessful. After a pioneering single treatment we now report the

  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......-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 ... 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...

  6. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    Science.gov (United States)

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

  7. The N-terminus of glycogen phosphorylase b is not required for activation by adenosine 5'-monophosphate.

    Science.gov (United States)

    Bigley, Andrew N; Reinhart, Gregory D

    2010-06-15

    The, so far unsuccessful, search for selective effective inhibitors of glycogen phosphorylase for the treatment of type II diabetes has made phosphorylase an active target of research for the past 20 years. Many crystallographic structures of phosphorylase are currently available to aid in this research. However, those structures have been interpreted, at least in part, on the basis of work conducted with a proteolytically derived form of phosphorylase that lacked the N-terminus (phosphorylase b'). It has been reported that phosphorylase b' shows no allostery, neither homotropic nor heterotropic. The original report on phosphorylase b' examined the allosteric characteristics over very narrow ranges of effector and substrate concentrations and reported the presence of proteolytic cleavages in addition to the removal of the N-terminus. We have applied molecular biological techniques to generate a truncate lacking the N-terminus with known primary structure, and we have established conditions for fully quantifying the allosteric effect of AMP on glycogen phosphorylase b. We report here for the first time the full thermodynamic effect of AMP on phosphorylase b. Our findings with a truncate lacking the N-terminus show that the effect of AMP binding does not depend on the N-terminus.

  8. Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP signaling to inhibit steroidogenesis in human granulosa cells

    Directory of Open Access Journals (Sweden)

    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 production, suggesting that the effect of leptin on steroidogenesis in granulosa cells is receptor dependent. In summary, leptin acts through the MAPK pathway to downregulate cAMP-induced StAR protein expression and progesterone production in immortalized human granulosa cells. These results suggest a possible mechanism by which gonadal steroidogenesis could be suppressed in obese women.

  9. A central role for neuronal adenosine 5'-monophosphate-activated protein kinase in cancer-induced anorexia.

    Science.gov (United States)

    Ropelle, Eduardo R; Pauli, José R; Zecchin, Karina G; Ueno, Mirian; de Souza, Cláudio T; Morari, Joseane; Faria, Marcel C; Velloso, Lício A; Saad, Mario J A; Carvalheira, José B C

    2007-11-01

    The pathogenesis of cancer anorexia is multifactorial and associated with disturbances of the central physiological mechanisms controlling food intake. However, the neurochemical mechanisms responsible for cancer-induced anorexia are unclear. Here we show that chronic infusion of 5-amino-4imidazolecarboxamide-riboside into the third cerebral ventricle and a chronic peripheral injection of 2 deoxy-d-glucose promotes hypothalamic AMP-activated protein kinase (AMPK) activation, increases food intake, and prolongs the survival of anorexic tumor-bearing (TB) rats. In parallel, the pharmacological activation of hypothalamic AMPK in TB animals markedly reduced the hypothalamic production of inducible nitric oxide synthase, IL-1beta, and TNF-alpha and modulated the expression of proopiomelanocortin, a hypothalamic neuropeptide that is involved in the control of energy homeostasis. Furthermore, the daily oral and intracerebroventricular treatment with biguanide antidiabetic drug metformin also induced AMPK phosphorylation in the central nervous system and increased food intake and life span in anorexic TB rats. Collectively, the findings of this study suggest that hypothalamic AMPK activation reverses cancer anorexia by inhibiting the production of proinflammatory molecules and controlling the neuropeptide expression in the hypothalamus, reflecting in a prolonged life span in TB rats. Thus, our data indicate that hypothalamic AMPK activation presents an attractive opportunity for the treatment of cancer-induced anorexia.

  10. Crystal chemistry of M{sup II}M′{sup IV}(PO{sub 4}){sub 2} double monophosphates

    Energy Technology Data Exchange (ETDEWEB)

    Bregiroux, Damien, E-mail: damien.bregiroux@upmc.fr [Sorbonne Universités, UPMC Univ Paris 06, CNRS, Collège de France, Laboratoire de Chimie de la Matière Condensée de Paris, 11 place Marcelin Berthelot, 75005 Paris (France); Popa, Karin [“Al.I. Cuza” University, Department of Chemistry, 11-Carol I Blvd., 700506 Iasi (Romania); Wallez, Gilles [Institut de Recherche de Chimie Paris (IRCP), CNRS – Chimie ParisTech – Paris Sciences et Lettres PSL UMR8247, 11 rue Pierre et Marie Curie, 75005 Paris (France); Sorbonne Universités, UPMC Univ Paris 06 (France)

    2015-10-15

    M{sup II}M′{sup IV}(PO{sub 4}){sub 2} compounds have been extensively studied for several decades for their potential applications in the field of several domains such as matrices for actinides conditioning, phosphors etc. In this paper, the relationships between composition and crystal structure of these compounds are established. A review of the various processes used for the synthesis of these compounds is also proposed, as well as their most reported properties. M{sup II}M′{sup IV}(PO{sub 4}){sub 2} structures stem from two different archetypes: the cheralite and the yavapaiite structures, with some exceptions that are also described in this article. The ratio of the cations radii appears to be the most relevant parameter. The high ratio between the ionic radii of the divalent and tetravalent cations in yavapaiite derivates results in the ordering of these cations into well-differentiated polyhedra whereas cheralite is the only non-ordered structure encountered for M{sup II}M′{sup IV}(PO{sub 4}){sub 2} compounds. - Graphical abstract: In this paper, the relationships between composition and crystal structure of M{sup II}M′{sup IV}(PO{sub 4}){sub 2} compounds are established. A review of the various processes used for the synthesis of these compounds is also proposed, as well as their most reported properties. - Highlights: • Crystal structure–composition relationships of MIIM′IV(PO4)2 compounds. • Review of the various processes used for the synthesis of these compounds. • Their most reported properties are described and discussed.

  11. Thermodynamic study of uranyl sorption onto lanthanum monophosphate (LaPO4) and magnetite (Fe3O4)

    International Nuclear Information System (INIS)

    Felix, Olivia

    2012-01-01

    The migration of radio-toxic elements in the geosphere is mainly regulated by chemical parameters which control the partitioning of the elements between mineral phases and aqueous solutions. Variation in temperature may affect the retention properties of a mineral surface and requires a careful investigation in order to understand the radionuclides behavior in the geosphere. In this way, the interaction mechanisms between uranium(VI) and two minerals (LaPO 4 and Fe 3 O 4 ) have been studied. In a first step, the monazite (LaPO 4 ) has been chosen as methodological solid in order to clearly define all the different stages needed to completely characterize the influence of temperature on the sorption phenomena. To reach that goal, three media, more or less complexants towards aqueous uranyl and the mineral surface, have been considered. Physico-chemical as well as surface acid-base properties of the solid surface have been studied by considering three electrolytes (NaClO 4 , NaNO 3 and Na 2 SO 4 ) and temperatures ranged from 25 C to 95 C. The point of zero charge has been found to be identical for perchlorate and nitrate media (pH PZC =2.1) but it was found to be one pK unit higher for the sulfate medium indicating a sorption of the background electrolyte ions. The reaction heats associated to the hydration of the solid have been measured by using microcalorimetry and the nature of the reactive surface sites has been determined by carrying out Time-Resolved Laser-Induced Fluorescence Spectroscopy (TRLFS). On the basis of these experimental constraints, the titration curves obtained for the monazite suspensions were fitted by using the Constant Capacitance Model and the 1-pK model was preferred to characterize the surface charge evolution, due to the limited number of adjustable parameters. The surface protonation constants being determined, the behavior of U(VI) towards the monazite surface in the three electrolytes has been investigated. On the basis of both U(VI) speciation in solution and the results of a structural study carrying out by using TRLFS together with calorimetric measurements, the sorption edges have been modeled and the corresponding sorption constants determined. Since these values take into account a wide number of experimental results (both structural and thermodynamical ones) they appear to be accurate and could be extrapolated more confidently to other physico-chemical conditions. The experimental approach being validated with the methodological solid, preliminary tests have been carried out to study uranyl sorption onto a second substrate, the magnetite, more relevant than monazite in the field of radionuclides migration in the geosphere. (author)

  12. Glycolytic pathway (GP), kreb's cycle (KC), and hexose monophosphate shunt (HMS) activity in myocardial subcellular fractions exposed to cannabinoids

    International Nuclear Information System (INIS)

    Watson, A.T.; Manno, B.R.; King, J.W.; Fowler, M.R.; Dempsey, C.A.; Manno, J.E.

    1986-01-01

    Delta-9-tetrahydrocannabinol (Δ 9 -THC), the primary psychoactive component of marihuana, and its active metabolite 11-hydroxy-Δ 9 -tetrahydrocannabinol (11-OH-Δ 9 -THC) have been reported to produce a direct cardiac depressant effect. Studies in isolated perfused rat hearts have indicated a decreased force of contraction (inotropic response) when Δ 9 -THC or 11-OH-Δ 9 -THC was administered in microgram amounts. The mechanism and site of action have not been explained or correlated with associated metabolic pathways. The purpose of this study was to investigate the effects of cannabinoids on major myocardial energy producing pathways, GP and KC, and a non-energy producing pathway, HMS. Cardiac ventricular tissue from male Sprague-Dawley rats (250-300 g) was excised and homogenized for subcellular fractionation. KC, GP and HMS activity was assayed in the appropriate fractions by measuring 14 CO 2 generation from 14 C-2-pyruvate, 14 C-6-glucose and 14 C-1-glucose respectively. Duplicate assays (n=8) were performed on tissue exposed to saline (control), empty liposomes (vehicle) and four doses each of Δ 9 -THC and 11-OH-Δ 9 -THC. Changes in metabolic activity and decreases in cardiac contractile performance may be associated

  13. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Kumar, S.

    1976-01-01

    Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi 80 cya + and phi 80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants

  14. Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways.

    Science.gov (United States)

    Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Hotta, Hak; Sada, Kiyonao

    2011-11-01

    Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

  15. 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...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  16. Modulation of agonist-induced inositol phosphate metabolism by cyclic adenosine 3',5'-monophosphate in adrenal glomerulosa cells

    International Nuclear Information System (INIS)

    Baukal, A.J.; Hunyady, L.; Balla, T.; Ely, J.A.; Catt, K.J.

    1990-01-01

    Activation of the cAMP messenger system was found to cause specific changes in angiotensin-II (All)-induced inositol phosphate production and metabolism in bovine adrenal glomerulosa cells. Pretreatment of [3H]inositol-labeled glomerulosa cells with 8-bromo-cAMP (8Br-cAMP) caused both short and long term changes in the inositol phosphate response to stimulation by All. Exposure to 8Br-cAMP initially caused dose-dependent enhancement (ED50 = 0.7 microM) of the stimulatory action of All (50 nM; 10 min) on the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and its immediate metabolites. This effect of 8Br-cAMP was also observed in permeabilized [3H]inositol-labeled glomerulosa cells in which degradation of Ins(1,4,5)P3 was inhibited, consistent with increased activity of phospholipase-C. Continued exposure to 8Br-cAMP for 5-16 h caused selective enhancement of the All-induced increases in D-myo-inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4] and myo-inositol 1,4,5,6-tetrakisphosphate. The long term effect of 8Br-cAMP on the 6-phosphorylated InsP4 isomers, but not the initial enhancement of Ins(1,4,5)P3 formation, was inhibited by cycloheximide. The characteristic biphasic kinetics of All-induced Ins(1,4,5)P3 formation were also changed by prolonged treatment with 8Br-cAMP to a monophasic response in which Ins(1,4,5)P3 increased rapidly and remained elevated during All stimulation. In permeabilized glomerulosa cells treated with 8Br-cAMP for 16 h, the conversion of D-myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] to Ins(1,3,4,6)P4 was consistently increased, whereas dephosphorylation of Ins(1,4,5)P3 to D-myo-inositol 1,4-bisphosphate and of D-myo-inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4)P3, was reduced

  17. Guanine limitation results in CodY-dependent and -independent alteration of Staphylococcus aureus physiology and gene expression.

    Science.gov (United States)

    King, Alyssa N; Borkar, Samiksha; Samuels, David J; Batz, Zachary; Bulock, Logan; Sadykov, Marat R; Bayles, Kenneth W; Brinsmade, Shaun R

    2018-04-30

    In Staphylococcus aureus , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on S. aureus physiology and CodY activity by constructing a guaA null mutant (Δ guaA ). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ guaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ guaA cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. Importance Staphylococcus aureus infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing

  18. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling

    KAUST Repository

    Turek, Ilona

    2016-03-05

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3′,5′-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. © 2016 Springer Science+Business Media Dordrecht

  19. The GDP-switched GAF domain of DcpA modulates the concerted synthesis/hydrolysis of c-di-GMP in Mycobacterium smegmatis.

    Science.gov (United States)

    Chen, Hui-Jie; Li, Na; Luo, Ye; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing; Li, Qiong

    2018-04-09

    The second messenger c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] plays a key role in bacterial growth, survival and pathogenesis, and thus its intracellular homeostasis should be finely maintained. Mycobacterium smegmatis encodes a GAF (mammalian c G MP-regulated phosphodiesterases, Anabaena a denylyl cyclases and Escherichia coli transcription activator F hlA) domain containing bifunctional enzyme DcpA ( d iguanylate c yclase and p hosphodiesterase A ) that catalyzes the synthesis and hydrolysis of c-di-GMP . Here, we found that M. smegmatis DcpA catalyzes the hydrolysis of c-di-GMP at a higher velocity, compared with synthetic activity, resulting in a sum reaction from the ultimate substrate GTP to the final product pGpG [5'-phosphoguanylyl-(3'-5')-guanosine]. Fusion with the N-terminal GAF domain enables the GGDEF (Gly-Gly-Asp-Glu-Phe) domain of DcpA to dimerize and accordingly gain synthetic activity. Screening of putative metabolites revealed that GDP is the ligand of the GAF domain. Binding of GDP to the GAF domain down-regulates synthetic activity, but up-regulates hydrolytic activity, which, in consequence, might enable a timely response to the transient accumulation of c-di-GMP at the stationary phase or under stresses. Combined with the crystal structure of the EAL (Glu-Ala-Leu) domain and the small-angle X-ray scattering data, we propose a putative regulatory model of the GAF domain finely tuned by the intracellular GTP/GDP ratio. These findings help us to better understand the concerted control of the synthesis and hydrolysis of c-di-GMP in M. smegmatis in various microenvironments. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  20. Nitric oxide-dependent vasodilation and Ca2+ signalling induced by erythrodiol in rat aorta

    Directory of Open Access Journals (Sweden)

    Fidèle Ntchapda

    2015-06-01

    Full Text Available Objective: To evaluate the pharmacological property of erythrodiol, a natural triterpenoid contained in propolis, as vasodilatory agent, and to determine its mechanism of action. Methods: Rats aortic rings were isolated and suspended in organ baths, and the effects of erythrodiol were studied by means of isometric tension recording experiments. Nitric oxide (NO was detected by ozone-induced chemiluminescence. The technique used to evaluate changes in intracellular Ca2+ concentration in intact endothelium was opened aortic ring and loaded with 16 µmol Fura-2/AM for 60 min at room temperature, washed and fixed by small pins with the luminal face up. In situ, ECs were visualized by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany equipped with a Zeiss×63 Achroplan objective (water immersion, 2.0 mm working distance, 0.9 numerical apertures. ECs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. Results: In aortic rings with intact endothelium pre-contracted with norepinephrine (10-4 mol/L, the addition of erythrodiol (10-8-10-4 mol/L induced vasorelaxation in a concentration-dependent manner; in endothelium-denuded rings, the relaxant response induced by erythrodiol was almost completely abolished suggesting that vasorelaxation was endothelium-dependent. They had almost no relaxant effect on depolarised or endothelium-denuded aortic segments. The relaxation was significantly attenuated by pre-treatment with the NO synthase inhibitor Nvnitro-L-arginine-methylester. Erythrodiol (10-4 mol/L was able to significantly increase NOx levels. This effect was completely abolished after removal of the vascular endothelium. Erythrodiol (100 µmol/L caused a slow, long-lasting increase in intracellular Ca2+ concentration. These results further supported the hypothesis that erythrodiol can induce activation of the NO/soluble guanylate cyclase/cyclic guanosine monophosphate pathway, as

  1. Vascular-Rheological Properties of Blood in Hemorrhagic Vasculitis Occurring in Childhood and Adulthood

    Directory of Open Access Journals (Sweden)

    V.V. Gerasymenko

    2016-11-01

    Full Text Available Background. As a result of the immune-inflammatory necrotic changes in the walls of arterioles and capillaries in patients with hemorrhagic vasculitis (HV Henoch — Schönlein endothelial dysfunction of vessels occurs, contributing to violations of blood rheological properties and microcirculation. These processes depend on the age of patients, and in cases of onset of the disease in childhood and adulthood are unknown. Objective: to study vascular and rheological properties of blood serum in HV and to compare the indices with different age of the debut of the pathological process in the groups of patients. Material and methods. The study included 174 patients with HV (83 % men and 17 % women. In 92 patients, the disease made its debut in childhood (on average in 12 years old, and in 82 — in the adult (on average in 25 years old. I, II and III degree of activity of pathological process are set at a ratio of 1 : 2 : 2. Indicators of vascular endothelial function were investigated by immune-enzyme analysis and the adsorption-rheological pro­perties of blood were assessed by computer tensiometry. Results. HV is accompanied by severe disorders of the blood vascular and rheological properties which are involved in the pathogenesis of lesions of skin (endothelin-1, surface tension, joints (only surface activity, kidney (prostacyclin, cyclic guanosine monophosphate and heart (endothelin-1, viscoelastic modulus. At that the integrated indicators of vascular endothelial function, viscoelastic, surface-active and relaxation characteristics of serum depend on the age of the patients in the beginning of the disease, the degree of activity of the pathological process, the clinical form of the disease course, necrotic-ulcerative and polymorphic variants of cutaneous vasculitis, and HV, transforming from juvenile, occurs with lower blood levels of endothelin-1, but with a higher concentration of thromboxane A2, cyclic guanosine monophosphate and

  2. Stress history increases alcohol intake in relapse: relation to phosphodiesterase 10A.

    Science.gov (United States)

    Logrip, Marian L; Zorrilla, Eric P

    2012-09-01

    Stressful experiences can result in elevated alcohol drinking, as exemplified in many individuals with post-traumatic stress disorder. However, how stress history, rather than acute stressors, influences alcohol intake remains uncertain. To model the protracted effects of past stress, male Wistar rats were subjected to light-cued footshock (stress history) or light cues alone (control) prior to acquisition of alcohol self-administration (1-hour sessions, fixed ratio 1-3, 100 µl of 10% v/v alcohol as reinforcer). Stress history did not alter mean alcohol intake during acquisition of self-administration, but it increased preference for the alcohol-paired lever over the inactive lever. Following an extinction period, rats with a history of stress exposure and low baseline alcohol intake showed a twofold elevation in alcohol self-administration, as compared with low-drinking rats with no stress history. Similar effects were not seen in rats self-administering 0.1% sucrose. Analysis of mRNA levels of phosphodiesterase 10A (PDE10A), a dual-specificity cyclic adenosine monophosphate and cyclic guanosine monophosphate hydrolyzing enzyme, showed that stress history increased Pde10a mRNA levels in the basolateral amygdala and, in low-drinking rats, the prelimbic prefrontal cortex (plPFC). Pde10a mRNA levels in the plPFC correlated directly with greater alcohol self-administration during the relapse-like phase, and greater BLA Pde10a mRNA levels correlated with increased ethanol preference after acquisition. The data demonstrate that stress history sensitizes otherwise low alcohol drinkers to consume more alcohol in a relapse-like situation and identify stress-induced neuroadaptations in amygdala and prefrontal cortical Pde10a expression as changes that may drive heightened alcohol intake and preference in susceptible individuals. © 2012 The Authors. Addiction Biology © 2012 Society for the Study of Addiction.

  3. The Effect of Alcohol Administration on the Corpus Cavernosum

    Directory of Open Access Journals (Sweden)

    See Min Choi

    2017-04-01

    Full Text Available Purpose: We studied the effects of alcohol administration on the corpus cavernosum (CC using an animal model. Materials and Methods: CC sections and the aortic ring of rabbits were used in an organ bath study. After acute alcohol administration, changes in blood alcohol concentration and electrical stimulation induced intracavernosal pressure/mean arterial pressure (ICP/MAP percentage were compared in rats. Cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP levels in the CC were measured using immunoassays. After chronic alcohol administration, ICP/MAP percentage, cAMP and cGMP were compared in rats. Histological changes were examined using the Masson trichrome stain and the Sircol collagen assay. Endothelial nitric oxide synthase (eNOS expression was examined using immunohistochemistry and Western blotting. Results: Alcohol relaxed the CC in a dose-dependent manner, and the relaxation response was suppressed when pretreated with propranolol, indomethacin, glibenclamide, and 4-aminopyridine. In rats with acute alcohol exposure, the cAMP level in the CC was significantly greater than was observed in the control group (p<0.05. In rats with chronic alcohol exposure, however, changes in cAMP and cGMP levels were insignificant, and the CC showed markedly smaller areas of smooth muscle, greater amounts of dense collagen (p<0.05. Immunohistochemical analysis of eNOS showed a less intense response, and western blotting showed that eNOS expression was significantly lower in this group (p<0.05. Conclusions: Acute alcohol administration activated the cAMP pathway with positive effects on erectile function. In contrast, chronic alcohol administration changed the ultrastructures of the CC and suppressed eNOS expression, thereby leading to erectile dysfunction.

  4. Acute effects of head-down tilt and hypoxia on modulators of fluid homeostasis

    Science.gov (United States)

    Whitson, P. A.; Cintron, N. M.; Pietrzyk, R. A.; Scotto, P.; Loeppky, J. A.

    1994-01-01

    In an effort to understand the interaction between acute postural fluid shifts and hypoxia on hormonal regulation of fluid homeostasis, the authors measured the responses to head-down tilt with and without acute exposure to normobaric hypoxia. Plasma atrial natriuretic peptide (ANP), cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), plasma aldosterone (ALD), and plasma renin activity (PRA) were measured in six healthy male volunteers who were exposed to a head-down tilt protocol during normoxia and hypoxia. The tilt protocol consisted of a 17 degrees head-up phase (30 minutes), a 28 degrees head-down phase (1 hour), and a 17 degrees head-up recovery period (2 hours, with the last hour normoxic in both experiments). Altitude equivalent to 14,828 ft was simulated by having the subjects breathe an inspired gas mixture with 13.9% oxygen. The results indicate that the postural fluid redistribution associated with a 60-minute head-down tilt induces the release of ANP and cGMP during both hypoxia and normoxia. Hypoxia increased cGMP, cAMP, ALD, and PRA throughout the protocol and significantly potentiated the increase in cGMP during head-down tilt. Hypoxia had no overall effect on the release of ANP, but appeared to attenuate the increase with head-down tilt. This study describes the acute effects of hypoxia on the endocrine response during fluid redistribution and suggests that the magnitude, but not the direction, of these changes with posture is affected by hypoxia.

  5. Relaxation effect of abacavir on rat basilar arteries.

    Directory of Open Access Journals (Sweden)

    Rachel Wai Sum Li

    Full Text Available The use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels.The relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5' nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate.Abacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5' nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries.Abacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may not be related to

  6. Studies on the mechanism of action of enterotoxin-induced fluid secretion in the gut

    International Nuclear Information System (INIS)

    Schirgi-Degen, A.

    1992-12-01

    The mechanism of action of Clostridium difficile enterotoxin A (CA), of Escherichia coli enterotoxin (STa) and of cholera toxin (CT), which are known to cause severe diarrhea, were studied in a preparation of ligated jejunal loops of anesthetized rats in vivo. The toxins were administered intraluminally. Pharmacological agents, which were tested for their potency to influence toxin-related effects, were administered subcutaneously. Net fluid transport was determined gravimetrically, prostaglandin (PG) E 2 -output into the lumen, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) contents in the mucosa were measured by radioimmunoassay, serotonin-(5-HT)-output into the lumen was determined by high performance liquid chromatography. The histopathological effects of CA and CT were examined by light- and scanning electron microscopy. All three toxins caused net fluid secretion (FS). 5-HT 2 -(ketanserin) and 5-HT 3 -receptor antagonists (tropisetron, ondansetron, granisetron) dose-dependently reduced or abolished CT- and STa-induced net FS, CA-induced net FS was not influenced. Indomethacin reduced CA-, CT- and STa-induced net FS. Elevation of PGE 2 -output occurred after exposure to CA and CT and was reduced by indomethacin. CA caused severe histopathological lesions and also CT time-dependently caused morphological changes, which may take part in the secretory response. It is concluded that 5-HT, using both 5-HT 2 - and 5-HT 3 -receptors, mediates CT- and STa, but not CA-induced FS. PGE 2 is involved in FS caused by all three toxins. CAMP and cGMP are presumedly no causative mediators of toxin-induced FS

  7. Correlative intravital imaging of cGMP signals and vasodilation in mice

    Directory of Open Access Journals (Sweden)

    Martin eThunemann

    2014-10-01

    Full Text Available Cyclic guanosine monophosphate (cGMP is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1 epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2 ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to ‘watch’ biochemistry, (patho physiology, and pharmacotherapy in the context of a living mammalian organism.

  8. Flavouring compounds in Indian potato snacks.

    Science.gov (United States)

    Raigond, Pinky; Singh, Brajesh; Dhulia, Akshita; Chopra, Shelly; Dutt, Som

    2015-12-01

    Market for processed potato products is rising day by day. Flavour plays important role in decision making by consumers due to their preferences for better tasting food. In potato and potato products, glutamic acid, aspartic acid, guanosine 5'-monophosphate (GMP) and adenosine 5'-monophosphate (AMP) are the major umami compounds which contribute towards flavour. Therefore, umami 5' nucleotides (AMP+GMP) were estimated from local potato products available as common fried products in the Indian markets and processed potato products being sold by the retailers. The analysis was also carried in raw, microwaved and pressure cooked tubers of forty seven Indian potato cultivars. Umami 5' nucleotide content ranged from 2.63 (Aloo seekh) to 8.26 μg/g FW (fried lachcha) in local potato products. In processed potato products, the content ranged from 2.72 μg/g FW (Smiles) to 14.75 μg/g FW (Aloo Bhujia). Along with aloo bhujia, umami 5' nucleotides were also high in dehydrated aloo lachcha (11.14 μg/g FW) and dehydrated potato chips (10.13 μg/g FW) and low in Smiles (2.72 μg/g FW) and Potato Shortz (3.40 μg/g FW). The study suggests that the potato products prepared solely from potato contained higher levels of umami 5' nucleotides compared to other products prepared by mixing potato with other cereals and vegetables. In Indian potato cultivars overall there was 14 % increase on microwave cooking and 31 % increase in flavouring compounds on pressure cooking. This type of study enabled in identifying better tasting cultivars for further product development and also to develop products with less addition of salt.

  9. Testosterone improves erectile function through inhibition of reactive oxygen species generation in castrated rats

    Directory of Open Access Journals (Sweden)

    Rui Li

    2016-05-01

    Full Text Available Testosterone is overwhelmingly important in regulating erectile physiology. However, the associated molecular mechanisms are poorly understood. The purpose of this study was to explore the effects and mechanisms of testosterone in erectile dysfunction (ED in castrated rats. Forty male Sprague-Dawley rats were randomized to four groups (control, sham-operated, castration and castration-with-testosterone-replacement. Reactive oxygen species (ROS production was measured by dihydroethidium (DHE staining. Erectile function was assessed by the recording of intracavernous pressure (ICP and mean arterial blood pressure (MAP. Protein expression levels were examined by western blotting. We found that castration reduced erectile function and that testosterone restored it. Nitric oxide synthase (NOS activity was decrease in the castrated rats, and testosterone administration attenuated this decrease (each p < 0.05. The testosterone, dihydrotestosterone, cyclic guanosine monophosphate (cGMP and cyclic adenosine monophosphate (cAMP concentrations were lower in the castrated rats, and testosterone restored these levels (each p < 0.05. Furthermore, the cyclooxygenase-2 (COX-2 and prostacyclin synthase (PTGIS expression levels and phospho-endothelial nitric oxide synthase (p-eNOS, Ser1177/endothelial nitric oxide synthase (eNOS ratio were reduced in the castrated rats compared with the controls (each p < 0.05. In addition, the p40phox and p67phox expression levels were increased in the castrated rats, and testosterone reversed these changes (each p < 0.05. Overall, our results demonstrate that testosterone ameliorates ED after castration by reducing ROS production and increasing the activity of the eNOS/cGMP and COX-2/PTGIS/cAMP signaling pathways.

  10. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway.

    Directory of Open Access Journals (Sweden)

    Peihong Dai

    2014-04-01

    Full Text Available Modified vaccinia virus Ankara (MVA is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs, which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs, but not in plasmacytoid dendritic cells (pDCs. Transcription factors IRF3 (IFN regulatory factor 3 and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1, are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase. MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1 and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

  11. Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Takashi Kawaguchi

    1999-01-01

    Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

  12. In vivo and in vitro animal investigation of the effect of a mixture of herbal extracts from Tribulus terrestris and Cornus officinalis on penile erection.

    Science.gov (United States)

    Kam, Sung Chul; Do, Jung Mo; Choi, Jae Hwi; Jeon, Byeong Tak; Roh, Gu Seob; Hyun, Jae Seog

    2012-10-01

    Herbal preparations have long been used as folk remedies for erectile dysfunction (ED). This study examined the effects of Tribulus terrestris and Cornus officinalis extracts on relaxation of the smooth muscle of the corpus cavernosum (CC), their mechanisms of action, and the effects of oral administration of a mixture of the herbal extracts on penile erection. The relaxation effects and the mechanisms of action of T. terrestris extract, C. officinalis extract, and the mixture of both extracts on the rabbit CC were investigated in an organ bath. To evaluate whether the relaxation response of the CC shown in an organ bath occurs in vivo, intracavernous pressure (ICP) was calculated in rats after oral administration for a month. Additionally, adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3', 5'-cyclic monophosphate (cGMP) in the CC were measured using immunoassay. Smooth muscle relaxation was expressed as the percent decrease in precontraction induced by phenylephrine. ICP was assessed in rats after the oral administration of a mixture of both extracts for 1 month and changes in cGMP and cAMP concentrations were measured based on the concentration of the mixture of both extracts. T. terrestris extract, C. officinalis extract, and the mixture of both extracts showed concentration-dependent relaxation effects of the CC. In both the endothelium-removed group and N(G)-nitro-L-arginine methyl ester pretreatment group, T. terrestris extract inhibited relaxation. ICP measured after oral administration of the extract mixture for a month was higher than that measured in the control group, and a significant increase in cAMP was observed in the mixture group. T. terrestris extract and C. officinalis extract exhibited concentration-dependent relaxation in an organ bath. In the in vivo study of the extract mixture, ICP and cAMP was significantly potentiated. Accordingly, the mixture of T. terrestris extract and C. officinalis extract may improve erectile function.

  13. Effects and Mechanism of Action of a Tribulus terrestris Extract on Penile Erection.

    Science.gov (United States)

    Do, Jungmo; Choi, Seemin; Choi, Jaehwi; Hyun, Jae Seog

    2013-03-01

    Tribulus terrestris has been used as an aphrodisiac. However, little is known about the effects and mechanism of action of T. terrestris on penile erection. Therefore, the effect of a T. terrestris extract and the mechanism of action of the extract on relaxation of the corpus cavernosum (CC) were investigated. The erectogenic effects of an oral preparation of the extract were also assessed. The relaxation effects and mechanism of action of the T. terrestris extract on rabbit CC were investigated in an organ bath. The intracavernous pressure (ICP) was calculated after oral administration of the extract for 1 month to evaluate whether the relaxation response of the CC shown in the organ bath occurred in vivo. Additionally, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in the CC by immunoassay. Smooth muscle relaxation was expressed as the percentage decrease in precontraction induced by phenylephrine. The ICP was also assessed in rats after oral administration of the extract for 1 month, and changes in concentrations of cGMP and cAMP were monitored. Concentration-dependent relaxation effects of the extract on the CC were detected in the organ bath study. Relaxation of the CC by the T. terrestris extract was inhibited in both an endothelium-removed group and an L-arginen methyl ester pretreatment group. The ICP measured after oral administration of the T. terrestris extract for 1 month was higher than that measured in the control group, and a significant increase in cAMP was observed in the T. terrestris extract group. The T. terrestris extract induced concentration-dependent relaxation of the CC in an organ bath. The mechanism included a reaction involving the nitric oxide/nitric oxide synthase pathway and endothelium of the CC. Moreover, in an in vivo study, the T. terrestris extract showed a significant concentration-dependent increase in ICP. Accordingly, the T. terrestris extract may improve erectile function.

  14. CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Patrick Oeckl

    Full Text Available BACKGROUND: The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP and cyclic guanosine-3',5'-monophosphate (cGMP are important second messengers and are potential biomarkers for Parkinson's disease (PD, amyotrophic lateral sclerosis (ALS and Creutzfeldt-Jakob disease (CJD. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS the cerebrospinal fluid (CSF concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15 had lower cAMP (-70% and cGMP (-55% concentrations in CSF compared with controls (n = 11. There was no difference in PD, PD dementia (PDD and ALS cases. Receiver operating characteristic (ROC curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC of 0.86 (cAMP and 0.85 (cGMP. We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92 and to 93% and 100% for the ratio tau/cAMP (AUC 0.99. CONCLUSIONS/SIGNIFICANCE: We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.

  15. Electromagnetic Field Devices and Their Effects on Nociception and Peripheral Inflammatory Pain Mechanisms.

    Science.gov (United States)

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

    2016-03-01

    Context • During cell-communication processes, endogenous and exogenous signaling affects normal and pathological developmental conditions. Exogenous influences, such as extra-low-frequency (ELF) electromagnetic fields (EMFs) have been shown to affect pain and inflammation by modulating G-protein coupling receptors (GPCRs), downregulating cyclooxygenase-2 (Cox-2) activity, and downregulating inflammatory modulators, such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) as well as the transcription factor nuclear factor kappa B (NF-κB). EMF devices could help clinicians who seek an alternative or complementary treatment for relief of patients chronic pain and disability. Objective • The research team intended to review the literature on the effects of EMFs on inflammatory pain mechanisms. Design • We used a literature search of articles published in PubMed using the following key words: low-frequency electromagnetic field therapy, inflammatory pain markers, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), opioid receptors, G-protein coupling receptors, and enzymes. Setting • The study took place at the Wake Forest School of Medicine in Winston-Salem, NC, USA. Results • The mechanistic pathway most often considered for the biological effects of EMF is the plasma membrane, across which the EMF signal induces a voltage change. Oscillating EMF exerts forces on free ions that are present on both sides of the plasma membrane and that move across the cell surface through transmembrane proteins. The ions create a forced intracellular vibration that is responsible for phenomena such as the influx of extracellular calcium (Ca2+) and the binding affinity of calmodulin (CaM), which is the primary transduction pathway to the secondary messengers, cAMP and cGMP, which have been found to influence inflammatory pain. Conclusions • An emerging body of evidence indicates the existence of a frequency

  16. Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-Terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis

    NARCIS (Netherlands)

    Houwing, C.J.; Huis in 't Veld, M.; Zuidema, D.; Graaff, de M.; Jaspars, E.M.J.

    2001-01-01

    Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural

  17. Measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine in cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry

    DEFF Research Database (Denmark)

    Weimann, Allan; Simonsen, Anja Hviid; Poulsen, Henrik E

    2018-01-01

    absolute concentrations varying by a factor >105 in healthy humans. This could indicate that there is a serious specificity problem in some of the methods. In this paper an isotope-dilution UPLC-MS/MS method with high specificity and sensitivity for the quantification of 8oxoGuo and 8oxodGuo in CSF...

  18. The mechanism of formation of (deoxy)guanosine adducts derived from peroxidase-catalyzed oxidation of the carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (SudanI)

    Czech Academy of Sciences Publication Activity Database

    Martínek, V.; Dračínský, Martin; Cvačka, Josef; Semanská, M.; Frei, E.; Stiborová, M.

    2009-01-01

    Roč. 276, Suppl. 1 (2009), s. 20-20 ISSN 1742-464X. [FEBS Congress /34/. 04.07.2009-09.07.2009, Praha] R&D Projects: GA AV ČR KJB400550903 Institutional research plan: CEZ:AV0Z40550506 Keywords : carcinogen * Sudan I metabolites * adducts Subject RIV: CC - Organic Chemistry

  19. Biological properties of novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles

    KAUST Repository

    Novak, Maria S.; Bü chel, Gabriel E.; Keppler, Bernhard K.; Jakupec, Michael A.

    2016-01-01

    Since the discovery that nitric oxide (NO) is a physiologically relevant molecule, there has been great interest in the use of metal nitrosyl compounds as antitumor pharmaceuticals. Particularly interesting are those complexes which can deliver NO to biological targets. Ruthenium- and osmium-based compounds offer lower toxicity compared to other metals and show different mechanisms of action as well as different spectra of activity compared to platinum-based drugs. Novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles were studied to elucidate their cytotoxicity and possible interactions with DNA. Apoptosis induction, changes of mitochondrial transmembrane potential and possible formation of reactive oxygen species were investigated as indicators of NO-mediated damage by flow cytometry. Results suggest that ruthenium- and osmium-nitrosyl complexes with the general formula (indazolium)[cis/trans-MCl4(NO)(1H-indazole)] have pronounced cytotoxic potency in cancer cell lines. Especially the more potent ruthenium complexes strongly induce apoptosis associated with depolarization of mitochondrial membranes, and elevated reactive oxygen species levels. Furthermore, a slight yet not unequivocal trend to accumulation of intracellular cyclic guanosine monophosphate attributable to NO-mediated effects was observed.

  20. Pathophysiology of anorexia in the cancer cachexia syndrome

    Science.gov (United States)

    Ezeoke, Chukwuemeka Charles; Morley, John E

    2015-01-01

    Anorexia is commonly present in persons with cancer and a major component of cancer cachexia. There are multiple causes of anorexia in cancer. Peripherally, these can be due to (i) substances released from or by the tumour, e.g. pro-inflammatory cytokines, lactate, and parathormone-related peptide; (ii) tumours causing dysphagia or altering gut function; (iii) tumours altering nutrients, e.g. zinc deficiency; (iv) tumours causing hypoxia; (v) increased peripheral tryptophan leading to increased central serotonin; or (vi) alterations of release of peripheral hormones that alter feeding, e.g. peptide tyrosine tyrosine and ghrelin. Central effects include depression and pain, decreasing the desire to eat. Within the central nervous system, tumours create multiple alterations in neurotransmitters, neuropeptides, and prostaglandins that modulate feeding. Many of these neurotransmitters appear to produce their anorectic effects through the adenosine monophosphate kinase/methylmalonyl coenzyme A/fatty acid system in the hypothalamus. Dynamin is a guanosine triphosphatase that is responsible for internalization of melanocortin 4 receptors and prostaglandin receptors. Dynamin is up-regulated in a mouse model of cancer anorexia. A number of drugs, e.g. megestrol acetate, cannabinoids, and ghrelin agonists, have been shown to have some ability to be orexigenic in cancer patients. PMID:26675762

  1. Investigation of the role of the NO-cGMP pathway on YC-1 and DEA/NO effects on thoracic aorta smooth muscle responses in a rat preeclampsia model.

    Science.gov (United States)

    Turgut, Nergiz Hacer; Temiz, Tijen Kaya; Turgut, Bülent; Karadas, Baris; Parlak, Mesut; Bagcivan, Ihsan

    2013-10-01

    The present study was designed to investigate the effects of YC-1, a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, a NO donor, on smooth muscle responses in the preeclampsia model with suramin-treated rats and on the levels of cyclic guanosine monophosphate (cGMP) of thoracic aorta rings isolated from term-pregnant rats. Rats of 2 groups, control group and suramin group, were given intraperitoneal injection of saline or suramin, respectively. Suramin injection caused increased blood pressure, protein in urine, and fetal growth retardation. Thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction and papaverine relaxation responses were similar. Relaxation responses of YC-1 and DEA/NO decreased in suramin group. In both groups in the presence of ODQ, a sGC inhibitor, the relaxation responses of YC-1 and DEA/NO decreased. The cGMP content was determined by radioimmunoassay technique. The content of cGMP in the suramin group decreased. In the presence of YC-1 and DEA/NO in both groups, cGMP content increased, but in ODQ-added groups, there was a significant decrease. We conclude that in preeclampsia, the decrease of relaxation responses and the decrease of cGMP content could be due to the reduction in stimulation of sGC and the decrease in cGMP levels.

  2. Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

    KAUST Repository

    Zhang, Gen; Wong, Yue-Him; Zhang, Yu; He, Li-sheng; Xu, Ying; Qian, Pei-Yuan

    2015-01-01

    Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/NO (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250 and 1000 μM) using a label-free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron-mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.

  3. Involvement of nitric oxide and ATP-sensitive potassium channels in the peripheral antinoceptive action of a tramadol-dexketoprofen combination in the formalin test.

    Science.gov (United States)

    Isiordia-Espinoza, Mario A; Pozos-Guillén, Amaury; Pérez-Urizar, José; Chavarría-Bolaños, Daniel

    2014-11-01

    Systemic coadministration of tramadol and dexketoprofen can produce antinociceptive synergism in animals. There has been only limited evaluation of this drug combination in the peripheral nervous system in terms of the antinociceptive interaction and its mechanisms. The aim of the present study was to evaluate the peripheral antinociceptive interaction between tramadol and dexketoprofen in the formalin test and the involvement of the nitric oxide (NO)-cyclic guanosine monophosphate pathway and ATP-sensitive K(+) channels. Different doses of tramadol or dexketoprofen were administered locally to the formalin-injured mouse paw and the antinociceptive effect evaluated. ED50 values were calculated for both drugs alone and in combination. Coadministration of tramadol and dexketoprofen produced an antinociceptive synergistic interaction during the second phase of the formalin test. Pretreatment with NO antagonists, including l-NG-nitroarginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, or the ATP-sensitive K(+) channel antagonist glibenclamide reversed the antinociceptive synergistic effect of the tramadol-dexketoprofen combination, suggesting that NO and ATP-sensitive K(+) channels were involved. © 2014 Wiley Periodicals, Inc.

  4. Biological properties of novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles

    KAUST Repository

    Novak, Maria S.

    2016-03-09

    Since the discovery that nitric oxide (NO) is a physiologically relevant molecule, there has been great interest in the use of metal nitrosyl compounds as antitumor pharmaceuticals. Particularly interesting are those complexes which can deliver NO to biological targets. Ruthenium- and osmium-based compounds offer lower toxicity compared to other metals and show different mechanisms of action as well as different spectra of activity compared to platinum-based drugs. Novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles were studied to elucidate their cytotoxicity and possible interactions with DNA. Apoptosis induction, changes of mitochondrial transmembrane potential and possible formation of reactive oxygen species were investigated as indicators of NO-mediated damage by flow cytometry. Results suggest that ruthenium- and osmium-nitrosyl complexes with the general formula (indazolium)[cis/trans-MCl4(NO)(1H-indazole)] have pronounced cytotoxic potency in cancer cell lines. Especially the more potent ruthenium complexes strongly induce apoptosis associated with depolarization of mitochondrial membranes, and elevated reactive oxygen species levels. Furthermore, a slight yet not unequivocal trend to accumulation of intracellular cyclic guanosine monophosphate attributable to NO-mediated effects was observed.

  5. The necrotic signal induced by mycophenolic acid overcomes apoptosis-resistance in tumor cells.

    Directory of Open Access Journals (Sweden)

    Gwendaline Guidicelli

    Full Text Available BACKGROUND: The amount of inosine monophosphate dehydrogenase (IMPDH, a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP, is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA-mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL-overexpressing cells. All tested cells remained sensitive to MPA-mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers. CONCLUSIONS/SIGNIFICANCE: These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

  6. New Nitric Oxide Donor NCX 1443: Therapeutic Effects on Pulmonary Hypertension in the SAD Mouse Model of Sickle Cell Disease.

    Science.gov (United States)

    Abid, Shariq; Kebe, Kanny; Houssaïni, Amal; Tomberli, Françoise; Marcos, Elisabeth; Bizard, Emilie; Breau, Marielle; Parpaleix, Aurelien; Tissot, Claire-Marie; Maitre, Bernard; Lipskaia, Larissa; Derumeaux, Genevieve; Bastia, Elena; Mekontso-Dessap, Armand; Adnot, Serge

    2018-05-01

    Nitric oxide (NO) donors may be useful for treating pulmonary hypertension (PH) complicating sickle cell disease (SCD), as endogenous NO is inactivated by hemoglobin released by intravascular hemolysis. Here, we investigated the effects of the new NO donor NCX1443 on PH in transgenic SAD mice, which exhibit mild SCD without severe hemolytic anemia. In SAD and wild-type (WT) mice, the pulmonary pressure response to acute hypoxia was similar and was abolished by 100 mg/kg NCX1443. The level of PH was also similar in SAD and WT mice exposed to chronic hypoxia (9% O2) alone or with SU5416 and was similarly reduced by daily NCX1443 gavage. Compared with WT mice, SAD mice exhibited higher levels of HO-1, endothelial NO synthase, and PDE5 but similar levels of lung cyclic guanosine monophosphate. Cultured pulmonary artery smooth muscle cells from SAD mice grew faster than those from WT mice and had higher PDE5 protein levels. Combining NCX1443 and a PDE5 inhibitor suppressed the growth rate difference between SAD and WT cells and induced a larger reduction in hypoxic PH severity in SAD than in WT mice. By amplifying endogenous protective mechanisms, NCX1443 in combination with PDE5 inhibition may prove useful for treating PH complicating SCD.

  7. 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; Ruzvidzo, Oziniel; Morse, Monique; Donaldson, Lara; Kwezi, Lusisizwe; Gehring, Christoph A

    2010-01-01

    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.

  8. Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

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    Tobias Fromme

    2018-02-01

    Full Text Available Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation. Keywords: Purine nucleotides, Uncoupling protein 1, Brown adipose tissue, Non-shivering thermogenesis, HILIC-MS/MS, Guanosine monophosphate reductase

  9. High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

    Science.gov (United States)

    Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

    2014-01-01

    Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

  10. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    Directory of Open Access Journals (Sweden)

    Mathieu Brochet

    2014-03-01

    Full Text Available Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission.

  11. Induction of expression of iNOS by N-nitrosodimethylamine (NDMA) in human leukocytes.

    Science.gov (United States)

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Jablonski, Jakub; Marcinczyk, Magdalena

    2009-01-01

    The aim of this study was to assess the influence of N-nitrosodimethylamine (NDMA) on expression of inducible nitric oxide synthase (iNOS), as well as production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC), and the participation of the p38 MAPK kinase in this process. Furthermore, the ability of neutrophils to release superoxide anion was determined. The influence of N-nitrosodimethylamine on iNOS expression was determined in isolated PMN and PBMC cells from peripheral blood of healthy individuals. The mononuclear cells showed higher sensitivity to NDMA. Moreover, cytotoxic effect of NDMA can be influenced in some way by the impact of this xenobiotic on nitric oxide and superoxide anion release from human leukocytes. Furthermore, increased generation of these radicals by human leukocytes suggest that neutrophils and mononuclear cells that are exposed to NDMA activity can play a key role in endogenous NDMA generation. However the relationship between iNOS expression and phospho-p38 MAPK in neutrophils and mononuclear cells shows that p38 MAPK pathway participates in induction of iNOS expression in the presence of NDMA.

  12. Pathophysiology of anorexia in the cancer cachexia syndrome.

    Science.gov (United States)

    Ezeoke, Chukwuemeka Charles; Morley, John E

    2015-12-01

    Anorexia is commonly present in persons with cancer and a major component of cancer cachexia. There are multiple causes of anorexia in cancer. Peripherally, these can be due to (i) substances released from or by the tumour, e.g. pro-inflammatory cytokines, lactate, and parathormone-related peptide; (ii) tumours causing dysphagia or altering gut function; (iii) tumours altering nutrients, e.g. zinc deficiency; (iv) tumours causing hypoxia; (v) increased peripheral tryptophan leading to increased central serotonin; or (vi) alterations of release of peripheral hormones that alter feeding, e.g. peptide tyrosine tyrosine and ghrelin. Central effects include depression and pain, decreasing the desire to eat. Within the central nervous system, tumours create multiple alterations in neurotransmitters, neuropeptides, and prostaglandins that modulate feeding. Many of these neurotransmitters appear to produce their anorectic effects through the adenosine monophosphate kinase/methylmalonyl coenzyme A/fatty acid system in the hypothalamus. Dynamin is a guanosine triphosphatase that is responsible for internalization of melanocortin 4 receptors and prostaglandin receptors. Dynamin is up-regulated in a mouse model of cancer anorexia. A number of drugs, e.g. megestrol acetate, cannabinoids, and ghrelin agonists, have been shown to have some ability to be orexigenic in cancer patients.

  13. Synthesis, Characterization and in Vitro Antitumor Activity of Platinum(II Oxalato Complexes Involving 7-Azaindole Derivatives as Coligands

    Directory of Open Access Journals (Sweden)

    Pavel Štarha

    2014-07-01

    Full Text Available The platinum(II oxalato complexes [Pt(ox(naza2] (1–3 were synthesized and characterized by elemental analysis (C, H, N, multinuclear NMR spectroscopy (1H, 13C, 15N, 195Pt and electrospray ionization mass spectrometry (ESI-MS; naza = 4-chloro-7-azaindole (4Claza; 1, 3-bromo-7-azaindole (3Braza; 2 or 4-bromo-7-azaindole (4Braza; 3. The prepared substances were screened for their in vitro antitumor activity on the osteosarcoma (HOS and breast adenocarcinoma (MCF7 human cancer cell lines, where 2 showed moderate antitumor effect (IC50 = 27.5 μM, and 18.3 μM, respectively. The complex 2 was further tested on a panel of six others human cancer cell lines, including the malignant melanoma (G361, cervix carcinoma (HeLa, ovarian carcinoma (A2780, cisplatin-resistant ovarian carcinoma (A2780R, lung carcinoma (A549 and prostate adenocarcinoma (LNCaP. This substance was found to be moderate antitumor effective against G361 (IC50 = 17.3 μM, HeLa (IC50 = 31.8 μM and A2780 (IC50 = 19.2 μM cell lines. The complex 2 was also studied by NMR for its solution stability and by ESI-MS experiments for its ability to interact with biomolecules, such as cysteine, glutathione or guanosine 5'-monophosphate.

  14. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

    KAUST Repository

    Kim, Jeong Joo

    2016-04-09

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

  15. cGAMP Quantification in Virus-Infected Human Monocyte-Derived Cells by HPLC-Coupled Tandem Mass Spectrometry.

    Science.gov (United States)

    Paijo, Jennifer; Kaever, Volkhard; Kalinke, Ulrich

    2017-01-01

    Upon virus infection, cells of the innate immune system such as dendritic cells and macrophages can mount type I interferon (IFN-I) responses that restrict viral dissemination. To inform host cells of virus infection, detection of cytosolic DNA is one important mechanism. Inappropriate sensing of endogenous DNA and subsequent induction of IFN-I responses can also cause autoimmunity, highlighting the need to tightly regulate DNA sensing. The cyclic GMP-AMP synthase (cGAS) was recently identified to be the major sensor of cytosolic DNA that triggers IFN-I expression. Upon DNA binding, cGAS synthesizes the second messenger cyclic guanosine-adenosine monophosphate (cGAMP) that induces IFN-I expression by the activation of the stimulator of interferon genes (STING). Notably, cGAMP does not only act in infected cells, but can also be relocated to noninfected bystander cells to there trigger IFN-I expression. Thus, direct quantification of cGAMP in cells of the innate immune system is an important approach to study where, when, and how DNA is sensed and IFN-I responses are induced. Here, we describe a method that allows specific quantification of cGAMP from extracts of virus-infected human myeloid cells by HPLC-coupled tandem mass spectrometry.

  16. Influence of gamma radiation and singlet oxygen on nucleic acid constituents

    International Nuclear Information System (INIS)

    Balland, Alain.

    1979-10-01

    The action of single oxygen on nucleosides proved to be extremely specific of deoxy-2' guanosine. The use of high performance liquid chromatography and spectrometric techniques (IR, mass and NMR) made it possible to isolate and characterise five main products of degradation. Ionizing radiations act mainly through radical species resulting from the radiolysis of water. The effects of the presence of DNA nucleosides in irradiated aqueous solutions of thymidine were investigated. It would appear, in these conditions, that the change in radio-sensitivity of thymidine in oxygenated solution can be explained essentially in terms of the competition of hydroxyl radicals. A study of the action of gamma rays on aqueous solutions of deoxy-2' guanilyl thymidine was carried out in the absence and presence of oxygen. The significant action of neutral radical species on the 'osidic' fragment explaining the break in the phosphodiester bond was noticed. The radio-induced modifications on the substrate were characterised indirectly by enzime hydrolysis (phosphodiesterasis). In an aerated aqueous solution, the monophosphate dinucleosides modified on the thymidine motive were identified by comparison with the substances obtained by synthesis. The characterisation of new substances and the study of synthetic ones required the use of NMR. Hence the configuration study of modified nucleosides was given much room [fr

  17. Evaluation of Pharmacokinetic and Pharmacodynamic Drug-Drug Interaction of Sacubitril/Valsartan (LCZ696) and Sildenafil in Patients With Mild-to-Moderate Hypertension.

    Science.gov (United States)

    Hsiao, H-L; Langenickel, T H; Petruck, J; Kode, K; Ayalasomayajula, S; Schuehly, U; Greeley, M; Pal, P; Zhou, W; Prescott, M F; Sunkara, G; Rajman, I

    2018-03-01

    Sacubitril/valsartan (LCZ696) is indicated for the treatment of patients with heart failure and reduced ejection fraction (HFrEF). Since patients with HFrEF may receive sacubitril/valsartan and sildenafil, both increasing cyclic guanosine monophosphate, the present study evaluated the pharmacokinetic and pharmacodynamic drug interaction potential between sacubitril/valsartan and sildenafil. In this open-label, three-period, single sequence study, patients with mild-to-moderate hypertension (153.8 ± 8.2 mmHg mean systolic blood pressure (SBP)) received a single dose of sildenafil 50 mg, sacubitril/valsartan 400 mg once daily for 5 days, and sacubitril/valsartan and sildenafil coadministration. When coadministered with sildenafil, the AUC and C max of valsartan decreased by 29% and 39%, respectively. Coadministration of sacubitril/valsartan and sildenafil resulted in a greater decrease in BP (-5/-4/-4 mmHg mean ambulatory SBP/DBP/MAP (mean arterial pressure)) than with sacubitril/valsartan alone. Both treatments were generally safe and well tolerated in this study; however, the additional BP reduction suggests that sildenafil should be administered cautiously in patients receiving sacubitril/valsartan. Unique identifier: NCT01601470. © 2017 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

  18. Evaluation of Pharmacokinetic and Pharmacodynamic Drug–Drug Interaction of Sacubitril/Valsartan (LCZ696) and Sildenafil in Patients With Mild‐to‐Moderate Hypertension

    Science.gov (United States)

    Langenickel, TH; Petruck, J; Kode, K; Ayalasomayajula, S; Schuehly, U; Greeley, M; Pal, P; Zhou, W; Prescott, MF; Sunkara, G; Rajman, I

    2017-01-01

    Sacubitril/valsartan (LCZ696) is indicated for the treatment of patients with heart failure and reduced ejection fraction (HFrEF). Since patients with HFrEF may receive sacubitril/valsartan and sildenafil, both increasing cyclic guanosine monophosphate, the present study evaluated the pharmacokinetic and pharmacodynamic drug interaction potential between sacubitril/valsartan and sildenafil. In this open‐label, three‐period, single sequence study, patients with mild‐to‐moderate hypertension (153.8 ± 8.2 mmHg mean systolic blood pressure (SBP)) received a single dose of sildenafil 50 mg, sacubitril/valsartan 400 mg once daily for 5 days, and sacubitril/valsartan and sildenafil coadministration. When coadministered with sildenafil, the AUC and Cmax of valsartan decreased by 29% and 39%, respectively. Coadministration of sacubitril/valsartan and sildenafil resulted in a greater decrease in BP (–5/–4/–4 mmHg mean ambulatory SBP/DBP/MAP (mean arterial pressure)) than with sacubitril/valsartan alone. Both treatments were generally safe and well tolerated in this study; however, the additional BP reduction suggests that sildenafil should be administered cautiously in patients receiving sacubitril/valsartan. Unique identifier: NCT01601470. PMID:28599060

  19. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  20. Comparison the effects of nitric oxide and spermidin pretreatment on alleviation of salt stress in chamomile plant (Matricaria recutita L.

    Directory of Open Access Journals (Sweden)

    Fazelian Nasrin

    2012-08-01

    Full Text Available Salt stress is an important environmental stress that produces reactive oxygen species in plants and causes oxidative injuries. In this investigation, salt stress reduced the shoot and root length, while increased the content of malondealdehyde, Hydrogen peroxide, and the activity of Ascorbate peroxidase andguaiacol peroxidase. Pretreatment of chamomile plants under salt stress with sodium nitroprussideand Spermidin caused enhancement of growth parameters and reduction of malondealdehyde and Hydrogen peroxide content. Pretreatment of plants with sodium nitroprusside remarkably increased Ascorbate peroxidase activity, while Spermidin pre-treatment significantly increased guaiacol peroxidase activity. Application of sodium nitroprusside or Spermidin with Methylene blue which is known to block cyclic guanosine monophosphate signaling pathway, reduced the protective effects of sodium nitroprussideand Spermidin in plants under salinity condition. The result of this study indicated that Methylene blue could partially and entirely abolish the protective effect of Nitric oxide on some physiological parameter. Methylene blue also has could reduce the alleviation effect of Spermidin on some of parameters in chamomile plant under salt stress, so with comparing the results of this study it seems that Spermidin probably acts through Nitric oxide pathway, but the use of 2-4- carboxyphenyl- 4,4,5,5- tetramethyl-imidazoline-1-oxyl-3-oxide is better to prove.

  1. Atrial natriuretic peptide stimulates salt secretion by shark rectal gland by releasing VIP

    Energy Technology Data Exchange (ETDEWEB)

    Silva, P.; Stoff, J.S.; Solomon, R.J.; Lear, S.; Kniaz, D.; Greger, R.; Epstein, F.H.

    1987-01-01

    Salt secretion by the isolated perfused rectal gland of the spiny dogfish shark, Squalus acanthias, is stimulated by synthetic rat atrial natriuretic peptide (ANP II) as well as extracts of shark heart, but not by 8-bromo-cyclic guanosine 5'-monophosphate. Cardiac peptides have no effect on isolated rectal gland cells or perfused tubules, suggesting that stimulation requires an intact gland. The stimulation of secretion by ANP II is eliminated by maneuvers that block neurotransmitter release. Cardiac peptides stimulate the release of vasoactive intestinal peptide (VIP), known to be present in rectal glands nerves, into the venous effluent of perfused glands in parallel with their stimulation of salt secretion, but the release of VIP induced by ANP II is prevented by perfusion with procaine. VIP was measured by radioimmunoassay. Cardiac peptides thus appear to regulate rectal gland secretion by releasing VIP from neural stores within the gland. It is possible that other physiological effects of these hormones might be explained by an action to enhanced local release of neurotransmitters.

  2. Gibberellic acid and cGMP-dependent transcriptional regulation in arabidopsis thaliana

    KAUST Repository

    Bastian, René

    2010-03-01

    An ever increasing amount of transcriptomic data and analysis tools provide novel insight into complex responses of biological systems. Given these resources we have undertaken to review aspects of transcriptional regulation in response to the plant hormone gibberellic acid (GA) and its second messenger guanosine 3\\',5\\'-cyclic monophosphate (cGMP) in Arabidopsis thaliana, both wild type and selected mutants. Evidence suggests enrichment of GA-responsive (GARE) elements in promoters of genes that are transcriptionally upregulated in response to cGMP but downregulated in a GA insensitive mutant (ga1-3). In contrast, in the genes upregulated in the mutant, no enrichment in the GARE is observed suggesting that GARE motifs are diagnostic for GA-induced and cGMP-dependent transcriptional upregulation. Further, we review how expression studies of GA-dependent transcription factors and transcriptional networks based on common promoter signatures derived from ab initio analyses can contribute to our understanding of plant responses at the systems level. © 2010 Landes Bioscience.

  3. Antinociceptive Activity of Methanol Extract of Muntingia calabura Leaves and the Mechanisms of Action Involved

    Directory of Open Access Journals (Sweden)

    M. H. Mohd. Sani

    2012-01-01

    Full Text Available Muntingia calabura L. (family Elaeocarpaceae has been traditionally used to relieve various pain-related ailments. The present study aimed to determine the antinociceptive activity of methanol extract of M. calabura leaves (MEMC and to elucidate the possible mechanism of antinociception involved. The in vivo chemicals (acetic acid-induced abdominal constriction and formalin-, capsaicin-, glutamate-, serotonin-induced paw licking test and thermal (hot plate test models of nociception were used to evaluate the extract antinociceptive activity. The extract (100, 250, and 500 mg/kg was administered orally 60 min prior to subjection to the respective test. The results obtained demonstrated that MEMC produced significant (P<0.05 antinociceptive response in all the chemical- and thermal-induced nociception models, which was reversed after pretreatment with 5 mg/kg naloxone, a non-selective opioid antagonist. Furthermore, pretreatment with L-arginine (a nitric oxide (NO donor, NG-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase (NOS, methylene blue (MB; an inhibitor of cyclic-guanosine monophosphate (cGMP pathway, or their combination also caused significant (P<0.05 change in the intensity of the MEMC antinociception. In conclusion, the MEMC antinociceptive activity involves activation of the peripheral and central mechanisms, and modulation via, partly, the opioid receptors and NO/cGMP pathway.

  4. Nitric oxide modulation of the basal ganglia circuitry: therapeutic implication for Parkinson's disease and other motor disorders.

    Science.gov (United States)

    Pierucci, Massimo; Galati, Salvatore; Valentino, Mario; Di Matteo, Vincenzo; Benigno, Arcangelo; Pitruzzella, Alessandro; Muscat, Richard; Di Giovanni, Giuseppe

    2011-11-01

    Several recent studies have emphasized a crucial role for the nitrergic system in movement control and the pathophysiology of the basal ganglia (BG). These observations are supported by anatomical evidence demonstrating the presence of nitric oxide synthase (NOS) in all the basal ganglia nuclei. In fact, nitrergic terminals have been reported to make synaptic contacts with both substantia nigra dopamine-containing neurons and their terminal areas such as the striatum, the globus pallidus and the subthalamus. These brain areas contain a high expression of nitric oxide (NO)-producing neurons, with the striatum having the greatest number, together with important NO afferent input. In this paper, the distribution of NO in the BG nuclei will be described. Furthermore, evidence demonstrating the nitrergic control of BG activity will be reviewed. The new avenues that the increasing knowledge of NO in motor control has opened for exploring the pathophysiology and pharmacology of Parkinson's disease and other movement disorders will be discussed. For example, inhibition of striatal NO/guanosine monophosphate signal pathway by phosphodiesterases seems to be effective in levodopa-induced dyskinesia. However, the results of experimental studies have to be interpreted with caution given the complexities of nitrergic signalling and the limitations of animal models. Nevertheless, the NO system represents a promising pharmacological intervention for treating Parkinson's disease and related disorders.

  5. Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

    Directory of Open Access Journals (Sweden)

    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.

  6. DNA catabolites in triathletes: effects of supplementation with an aronia-citrus juice (polyphenols-rich juice).

    Science.gov (United States)

    García-Flores, Libia Alejandra; Medina, Sonia; Cejuela-Anta, Roberto; Martínez-Sanz, José Miguel; Abellán, Ángel; Genieser, Hans-Gottfried; Ferreres, Federico; Gil-Izquierdo, Ángel

    2016-04-01

    In this study we analyzed whether our aronia-citrus juice (ACJ, the composition is based on a mixture of 95% citrus juice with 5% of Aronia melanocarpa juice), rich in polyphenols, and physical exercise had an effect on seven catabolites of DNA identified in plasma and on a urine isoprostane (8-iso-PGF2α). Sixteen elite triathletes on a controlled diet for triathlon training (45 days) were used in this clinical trial. Our results show a decrease in the 8-hydroxy-2'-deoxyguanosine concentration due to chronic physical exercise. The ACJ intake and physical exercise maintained the guanosine-3',5'-cyclic monophosphate plasmatic concentrations and decreased the concentration of 8-hydroxyguanine as well as urinary values of 8-iso-PGF2α. Finally, we observed a significant increase in the 8-nitroguanosine levels in triathletes after ACJ intake, compared to the placebo stage. It is concluded that the combination of the intake of ACJ, rich in polyphenolic compounds, with adequate training was able to influence the plasmatic and urinary values of oxidative stress biomarkers. This suggests a positive effect on the oxidative damage and potential associations with DNA repair mechanisms.

  7. Sildenafil citrate (Viagra) enhances vasodilatation by atrial natriuretic peptide in normal dogs.

    Science.gov (United States)

    Ishikura, Fuminobu; Beppu, Shintaro; Asanuma, Toshihiko; Seward, James B; Khandheria, Bijoy K

    2007-12-01

    Sildenafil citrate (Viagra) is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5, which might enhance the vasorelaxant and natriuretic actions of atrial natriuretic peptide (ANP) in patients with heart failure. The objective of this study was to examine the combined effect of Viagra on hemodynamic changes during infusion of exogenous ANP. Healthy male beagles were used to assess systemic blood pressure, pulmonary artery pressure (PAP), and plasma levels of cGMP. After hemodynamic variables were measured, 0.1 microg.kg(-1).min(-1) of ANP was given during this study. One hour after initiating infusion of ANP, 2 mg/kg of sildenafil citrate or vehicle was given orally via a nasogastric tube. Hemodynamic changes were measured before and 1 h after these administrations. Mean systemic and PAP decreased during infusion of ANP, and further decreased after sildenafil citrate administration, however, mean systemic blood pressure decreased within 10 mmHg. Plasma levels of cGMP also increased after sildenafil citrate administration. In normal dogs, sildenafil citrate enhances the vasodilator effect of ANP by increasing the cGMP level, however, the concomitant use of sildenafil citrate with ANP will not induce severe hypotension.

  8. Sildenafil Prevents Apoptosis of Human First-Trimester Trophoblast Cells Exposed to Oxidative Stress

    Science.gov (United States)

    Bolnick, Jay M.; Kilburn, Brian A.; Bolnick, Alan D.; Diamond, Michael P.; Singh, Manvinder; Hertz, Michael; Dai, Jing

    2015-01-01

    Human first-trimester trophoblast cells proliferate at low O2, but survival is compromised by oxidative stress, leading to uteroplacental insufficiency. The vasoactive drug, sildenafil citrate (Viagra, Sigma, St Louis, Missouri), has proven useful in reducing adverse pregnancy outcomes. An important biological function of this pharmaceutical is its action as an inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterase type 5 activity, which suggests that it could have beneficial effects on trophoblast survival. To investigate whether sildenafil can prevent trophoblast cell death, human first-trimester villous explants and the HTR-8/SVneo cytotrophoblast cell line were exposed to hypoxia and reoxygenation (H/R) to generate oxidative stress, which induces apoptosis. Apoptosis was optimally inhibited during H/R by 350 ng/mL sildenafil. Sildenafil-mediated survival was reversed by l-NG-nitro-l-arginine methyl ester hydrochloride or cGMP antagonist, indicating a dependence on both nitric oxide (NO) and cGMP. Indeed, either a cGMP agonist or an NO generator was cytoprotective independent of sildenafil. These findings suggest a novel intervention route for patients with recurrent pregnancy loss or obstetrical placental disorders. PMID:25431453

  9. Sildenafil Protects against Myocardial Ischemia-Reperfusion Injury Following Cardiac Arrest in a Porcine Model: Possible Role of the Renin-Angiotensin System

    Science.gov (United States)

    Wang, Guoxing; Zhang, Qian; Yuan, Wei; Wu, Junyuan; Li, Chunsheng

    2015-01-01

    Sildenafil, a phosphodiesterase-5 inhibitor sold as Viagra, is a cardioprotector against myocardial ischemia/reperfusion (I/R) injury. Our study explored whether sildenafil protects against I/R-induced damage in a porcine cardiac arrest and resuscitation (CAR) model via modulating the renin-angiotensin system. Male pigs were randomly divided to three groups: Sham group, Saline group, and sildenafil (0.5 mg/kg) group. Thirty min after drug infusion, ventricular fibrillation (8 min) and cardiopulmonary resuscitation (up to 30 min) was conducted in these animals. We found that sildenafil ameliorated the reduced cardiac function and improved the 24-h survival rate in this model. Sildenafil partly attenuated the increases of plasma angiotensin II (Ang II) and Ang (1–7) levels after CAR. Sildenafil also decreased apoptosis and Ang II expression in myocardium. The increases of expression of angiotensin-converting-enzyme (ACE), ACE2, Ang II type 1 receptor (AT1R), and the Ang (1–7) receptor Mas in myocardial tissue were enhanced after CAR. Sildenafil suppressed AT1R up-regulation, but had no effect on ACE, ACE2, and Mas expression. Sildenafilfurther boosted the upregulation of endothelial nitric oxide synthase (eNOS), cyclic guanosine monophosphate (cGMP) and inducible nitric oxide synthase(iNOS). Collectively, our results suggest that cardioprotection of sildenafil in CAR model is accompanied by an inhibition of Ang II-AT1R axis activation. PMID:26569234

  10. Sildenafil Protects against Myocardial Ischemia-Reperfusion Injury Following Cardiac Arrest in a Porcine Model: Possible Role of the Renin-Angiotensin System

    Directory of Open Access Journals (Sweden)

    Guoxing Wang

    2015-11-01

    Full Text Available Sildenafil, a phosphodiesterase-5 inhibitor sold as Viagra, is a cardioprotector against myocardial ischemia/reperfusion (I/R injury. Our study explored whether sildenafil protects against I/R-induced damage in a porcine cardiac arrest and resuscitation (CAR model via modulating the renin-angiotensin system. Male pigs were randomly divided to three groups: Sham group, Saline group, and sildenafil (0.5 mg/kg group. Thirty min after drug infusion, ventricular fibrillation (8 min and cardiopulmonary resuscitation (up to 30 min was conducted in these animals. We found that sildenafil ameliorated the reduced cardiac function and improved the 24-h survival rate in this model. Sildenafil partly attenuated the increases of plasma angiotensin II (Ang II and Ang (1–7 levels after CAR. Sildenafil also decreased apoptosis and Ang II expression in myocardium. The increases of expression of angiotensin-converting-enzyme (ACE, ACE2, Ang II type 1 receptor (AT1R, and the Ang (1–7 receptor Mas in myocardial tissue were enhanced after CAR. Sildenafil suppressed AT1R up-regulation, but had no effect on ACE, ACE2, and Mas expression. Sildenafilfurther boosted the upregulation of endothelial nitric oxide synthase (eNOS, cyclic guanosine monophosphate (cGMP and inducible nitric oxide synthase(iNOS. Collectively, our results suggest that cardioprotection of sildenafil in CAR model is accompanied by an inhibition of Ang II-AT1R axis activation.

  11. Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

    KAUST Repository

    Zhang, Gen

    2015-08-28

    Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/NO (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250 and 1000 μM) using a label-free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron-mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.

  12. cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

    Science.gov (United States)

    Wincott, Charlotte M; Abera, Sinedu; Vunck, Sarah A; Tirko, Natasha; Choi, Yoon; Titcombe, Roseann F; Antoine, Shannon O; Tukey, David S; DeVito, Loren M; Hofmann, Franz; Hoeffer, Charles A; Ziff, Edward B

    2014-10-01

    Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response. Published by Elsevier Inc.

  13. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    International Nuclear Information System (INIS)

    Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

    2014-01-01

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress

  14. Atrial natriuretic peptide stimulates salt secretion by shark rectal gland by releasing VIP

    International Nuclear Information System (INIS)

    Silva, P.; Stoff, J.S.; Solomon, R.J.; Lear, S.; Kniaz, D.; Greger, R.; Epstein, F.H.

    1987-01-01

    Salt secretion by the isolated perfused rectal gland of the spiny dogfish shark, Squalus acanthias, is stimulated by synthetic rat atrial natriuretic peptide (ANP II) as well as extracts of shark heart, but not by 8-bromo-cyclic guanosine 5'-monophosphate. Cardiac peptides have no effect on isolated rectal gland cells or perfused tubules, suggesting that stimulation requires an intact gland. The stimulation of secretion by ANP II is eliminated by maneuvers that block neurotransmitter release. Cardiac peptides stimulate the release of vasoactive intestinal peptide (VIP), known to be present in rectal glands nerves, into the venous effluent of perfused glands in parallel with their stimulation of salt secretion, but the release of VIP induced by ANP II is prevented by perfusion with procaine. VIP was measured by radioimmunoassay. Cardiac peptides thus appear to regulate rectal gland secretion by releasing VIP from neural stores within the gland. It is possible that other physiological effects of these hormones might be explained by an action to enhanced local release of neurotransmitters

  15. Liquid microjet synchrotron-radiation spectroscopy for biomolecules in water solution 2

    International Nuclear Information System (INIS)

    Shimada, Hiroyuki; Ukai, Masatoshi

    2014-01-01

    A new spectroscopic research of radiation induced damage on DNA and its constituent molecules is proposed, which is made possible using a liquid microjet technique for bio-solution under vacuum in combination with synchrotron-radiation aided site-selective excitation. The latter part of the proposal article describes the present state of research on the selective primary radiation interaction by looking at base moieties of nucleotides. X-ray absorption near edge structure (XANES) spectra at energies around the nitrogen K-edge for nucleotides, adenosine-5'-monophosphate (AMP), guanosine-5'-monophosophate (GMP), cytidine-5'-monophosophate (CMP), and adenosine-5'-triphosphate (ATP) in aqueous solutions are presented. Selective excitation of a base moiety using a synchrotron radiation allows us to investigate the interaction of the base moiety with water solvent. We discuss the change of spectral character of XANES which reveals to the structural change of the base moiety under different pH environmental condition of water solution. Through the present research a scope for cooperative direct and indirect primary radiation effects is given. (author)

  16. Taurine Supplementation Improves Erectile Function in Rats with Streptozotocin-induced Type 1 Diabetes via Amelioration of Penile Fibrosis and Endothelial Dysfunction.

    Science.gov (United States)

    Ruan, Yajun; Li, Mingchao; Wang, Tao; Yang, Jun; Rao, Ke; Wang, Shaogang; Yang, Weiming; Liu, Jihong; Ye, Zhangqun

    2016-05-01

    For patients with diabetes, erectile dysfunction (ED) is common and greatly affects quality of life. However, these patients often exhibit a poor response to first-line oral phosphodiesterase type 5 inhibitors. To investigate whether taurine, a sulfur-containing amino acid, affects diabetic ED (DED). Type 1 diabetes mellitus was induced in male rats by using streptozotocin. After 12 weeks, an apomorphine test was conducted to confirm DED. Only rats with DED were administered taurine or vehicle for 4 weeks. Age-matched nondiabetic rats were administered saline intraperitoneally for 4 weeks. Erectile function was evaluated by electrical stimulation of the cavernous nerve. Histologic and molecular alterations of the corpus cavernosum also were analyzed. Erectile function was significantly reduced in the diabetic rats compared with in the nondiabetic rats, and was improved in the diabetic rats treated with taurine. The corpus cavernosum of the rats with DED exhibited severe fibrosis and decreased smooth muscle content. Deposition of extracellular matrix proteins was increased in the diabetic rats, while expression of endothelial nitric oxide synthase/cyclic guanosine monophosphate/nitric oxide pathway-related proteins was reduced. Taurine supplementation ameliorated erectile response as well as histologic and molecular alterations. Taurine supplementation improves erectile function in rats with DED probably by potential antifibrotic activity. This finding provides evidence for a potential new therapy for DED. Copyright © 2016 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

  17. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    Directory of Open Access Journals (Sweden)

    Lorena Olivares-González

    Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  18. Food-Grade Synthesis of Maillard-Type Taste Enhancers Using Natural Deep Eutectic Solvents (NADES).

    Science.gov (United States)

    Kranz, Maximilian; Hofmann, Thomas

    2018-01-28

    The increasing demand for healthier food products, with reduced levels of table salt, sugar, and mono sodium glutamate, reinforce the need for novel taste enhancers prepared by means of food-grade kitchen-type chemistry. Although several taste modulating compounds have been discovered in processed foods, their Maillard-type ex food production is usually not exploited by industrial process reactions as the yields of target compounds typically do not exceed 1-2%. Natural deep eutectic solvents (NADES) are reported for the first time to significantly increase the yields of the taste enhancers 1-deoxy-ᴅ-fructosyl-N-β-alanyl-ʟ-histidine (49% yield), N-(1-methyl-4-oxoimidazolidin-2-ylidene) aminopropionic acid (54% yield) and N²-(1-carboxyethyl) guanosine 5'-monophosphate (22% yield) at low temperature (80-100 °C) within a maximum reaction time of 2 h. Therefore, NADES open new avenues to a "next-generation culinary chemistry" overcoming the yield limitations of traditional Maillard chemistry approaches and enable a food-grade Maillard-type generation of flavor modulators.

  19. Sildenafil Can Affect Innate and Adaptive Immune System in Both Experimental Animals and Patients.

    Science.gov (United States)

    Kniotek, Monika; Boguska, Agnieszka

    2017-01-01

    Sildenafil, a type 5 phosphodiesterase inhibitor (PDE5-I), is primarily used for treating erectile dysfunction. Sildenafil inhibits the degradation of cyclic guanosine monophosphate (cGMP) by competing with cGMP for binding site of PDE5. cGMP is a secondary messenger activating protein kinases and a common regulator of ion channel conductance, glycogenolysis, and cellular apoptosis. PDE5 inhibitors (PDE-Is) found application in cardiology, nephrology, urology, dermatology, oncology, and gynecology. Positive result of sildenafil treatment is closely connected with its immunomodulatory effects. Sildenafil influences angiogenesis, platelet activation, proliferation of regulatory T cells, and production of proinflammatory cytokines and autoantibodies. Sildenafil action in humans and animals appears to be different. Surprisingly, it also acts differently in males and females organisms. Although the immunomodulatory effects of PDE5 inhibitors appear to be promising, none of them reached the point of being tested in clinical trials. Data on the influence of selective PDE5-Is on the human immune system are limited. The main objective of this review is to discuss the immunomodulatory effects of sildenafil in both patients and experimental animals. This is the first review of the current state of knowledge about the effects of sildenafil on the immune system.

  20. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

    Directory of Open Access Journals (Sweden)

    Robert W Moon

    2009-09-01

    Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.

  1. Bacterial Biofilm Control by Perturbation of Bacterial Signaling Processes

    Directory of Open Access Journals (Sweden)

    Tim Holm Jakobsen

    2017-09-01

    Full Text Available The development of effective strategies to combat biofilm infections by means of either mechanical or chemical approaches could dramatically change today’s treatment procedures for the benefit of thousands of patients. Remarkably, considering the increased focus on biofilms in general, there has still not been invented and/or developed any simple, efficient and reliable methods with which to “chemically” eradicate biofilm infections. This underlines the resilience of infective agents present as biofilms and it further emphasizes the insufficiency of today’s approaches used to combat chronic infections. A potential method for biofilm dismantling is chemical interception of regulatory processes that are specifically involved in the biofilm mode of life. In particular, bacterial cell to cell signaling called “Quorum Sensing” together with intracellular signaling by bis-(3′-5′-cyclic-dimeric guanosine monophosphate (cyclic-di-GMP have gained a lot of attention over the last two decades. More recently, regulatory processes governed by two component regulatory systems and small non-coding RNAs have been increasingly investigated. Here, we review novel findings and potentials of using small molecules to target and modulate these regulatory processes in the bacterium Pseudomonas aeruginosa to decrease its pathogenic potential.

  2. Plant natriuretic peptides: Systemic regulators of plant homeostasis and defense that can affect cardiomyoblasts

    KAUST Repository

    Gehring, Christoph A.

    2010-09-01

    Immunologic evidence has suggested the presence of biologically active natriuretic peptide (NPs) hormones in plants because antiatrial NP antibodies affinity purify biologically active plant NPs (PNP). In the model plant, an Arabidopsis thaliana PNP (AtPNP-A) has been identified and characterized. AtPNP-A belongs to a novel class of molecules that share some similarity with the cell wall loosening expansins but do not contain the carbohydrate-binding wall anchor thus suggesting that PNPs and atrial natriuretic peptides are heterologs. AtPNP-A acts systemically, and this is consistent with its localization in the apoplastic extracellular space and the conductive tissue. Furthermore, AtPNP-A signals via the second messenger cyclic guanosine 3′,5′-monophosphate and modulates ion and water transport and homeostasis. It also plays a critical role in host defense against pathogens. AtPNP-A can be classified as novel paracrine plant hormone because it is secreted into the apoplastic space in response to stress and can enhance its own expression. Interestingly, purified recombinant PNP induces apo-ptosis in a dose-dependent manner and was most effective on cardiac myoblast cell lines. Because PNP is mimicking the effect of ANP in some instances, PNP may prove to provide useful leads for development of novel therapeutic NPs. Copyright © 2013 by The American Federation for Medical Research.

  3. Role of the hypothalamic pituitary adrenal axis in the control of the response to stress and infection

    Directory of Open Access Journals (Sweden)

    McCann S.M.

    2000-01-01

    Full Text Available The release of adrenocorticotropin (ACTH from the corticotrophs is controlled principally by vasopressin and corticotropin-releasing hormone (CRH. Oxytocin may augment the release of ACTH under certain conditions, whereas atrial natriuretic peptide acts as a corticotropin release-inhibiting factor to inhibit ACTH release by direct action on the pituitary. Glucocorticoids act on their receptors within the hypothalamus and anterior pituitary gland to suppress the release of vasopressin and CRH and the release of ACTH in response to these neuropeptides. CRH neurons in the paraventricular nucleus also project to the cerebral cortex and subcortical regions and to the locus ceruleus (LC in the brain stem. Cortical influences via the limbic system and possibly the LC augment CRH release during emotional stress, whereas peripheral input by pain and other sensory impulses to the LC causes stimulation of the noradrenergic neurons located there that project their axons to the CRH neurons stimulating them by alpha-adrenergic receptors. A muscarinic cholinergic receptor is interposed between the alpha-receptors and nitric oxidergic interneurons which release nitric oxide that activates CRH release by activation of cyclic guanosine monophosphate, cyclooxygenase, lipoxygenase and epoxygenase. Vasopressin release during stress may be similarly mediated. Vasopressin augments the release of CRH from the hypothalamus and also augments the action of CRH on the pituitary. CRH exerts a positive ultrashort loop feedback to stimulate its own release during stress, possibly by stimulating the LC noradrenergic neurons whose axons project to the paraventricular nucleus to augment the release of CRH.

  4. In Silico Design of Human IMPDH Inhibitors Using Pharmacophore Mapping and Molecular Docking Approaches

    Directory of Open Access Journals (Sweden)

    Rui-Juan Li

    2015-01-01

    Full Text Available Inosine 5′-monophosphate dehydrogenase (IMPDH is one of the crucial enzymes in the de novo biosynthesis of guanosine nucleotides. It has served as an attractive target in immunosuppressive, anticancer, antiviral, and antiparasitic therapeutic strategies. In this study, pharmacophore mapping and molecular docking approaches were employed to discover novel Homo sapiens IMPDH (hIMPDH inhibitors. The Güner-Henry (GH scoring method was used to evaluate the quality of generated pharmacophore hypotheses. One of the generated pharmacophore hypotheses was found to possess a GH score of 0.67. Ten potential compounds were selected from the ZINC database using a pharmacophore mapping approach and docked into the IMPDH active site. We find two hits (i.e., ZINC02090792 and ZINC00048033 that match well the optimal pharmacophore features used in this investigation, and it is found that they form interactions with key residues of IMPDH. We propose that these two hits are lead compounds for the development of novel hIMPDH inhibitors.

  5. 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.

  6. Loss of CO2 sensing by the olfactory system of CNGA3 knockout mice

    Directory of Open Access Journals (Sweden)

    Jinlong HAN, Minmin LUO

    2010-12-01

    Full Text Available Atmospheric CO2 can signal the presence of food, predators or environmental stress and trigger stereotypical behaviors in both vertebrates and invertebrates. Recent studies have shown that the necklace olfactory system in mice sensitively detects CO2 in the air. Olfactory CO2 neurons are believed to rely on cyclic guanosine monophosphate (cGMP as the key second messenger; however, the specific ion channel underlying CO­2 responses remains unclear. Here we show that CO2-evoked neuronal and behavioral responses require cyclic nucleotide-gated (CNG channels consisting of the CNGA3 subunit. Through Ca2+-imaging, we found that CO2-triggered Ca2+ influx was abolished in necklace olfactory sensory neurons (OSNs of CNGA3-knockout mice. Olfactory detection tests using a Go/No-go paradigm showed that these knockout mice failed to detect 0.5% CO2. Thus, sensitive detection of atmospheric CO2 depends on the function of CNG channels consisting of the CNGA3 subunit in necklace OSNs. These data support the important role of the necklace olfactory system in CO2 sensing and extend our understanding of the signal transduction pathway mediating CO2 detection in mammals [Current Zoology 56 (6: 793–799, 2010].

  7. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

    KAUST Repository

    Kim, Jeong  Joo; Lorenz, Robin; Arold, Stefan T.; Reger, Albert  S.; Sankaran, Banumathi; Casteel, Darren  E.; Herberg, Friedrich  W.; Kim, Choel

    2016-01-01

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

  8. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    Science.gov (United States)

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

  9. The protective effects of sildenafil in acute lung injury in a rat model of severe scald burn: A biochemical and histopathological study.

    Science.gov (United States)

    Gokakin, Ali Kagan; Deveci, Koksal; Kurt, Atilla; Karakus, Boran Cihat; Duger, Cevdet; Tuzcu, Mehmet; Topcu, Omer

    2013-09-01

    Severe burn induces biochemical mediators such as reactive oxygen species that leads to lipid peroxidation which may have a key role in formation of acute lung injury (ALI). Sildenafil is a selective and potent inhibitor of cyclic guanosine monophosphate specific phosphodiesterase-5. Sildenafil preserves alveolar growth, angiogenesis, reduces inflammation and airway reactivity. The purpose of the present study was to evaluate the effects of different dosages of sildenafil in ALI due to severe scald burn in rats. Twenty-four rats were subjected to 30% total body surface area severe scald injury and were randomly divided into three equal groups as follow: control, 10 and 20mg/kg sildenafil groups. Levels of malondialdehyde (MDA), activities of glutathione peroxidase (Gpx), catalase (Cat), total oxidative stress (TOS), and total antioxidative capacity (TAC) were measured in both tissues and serums. Oxidative stress index (OSI) was calculated. A semi-quantitative scoring system was used for the evaluation of histopatological findings. Sildenafil increased Gpx, Cat, TAC and decreased MDA, TOS and OSI. Sildenafil decreased inflammation scores in lungs. Our results reveal that sildenafil is protective against scald burn related ALI by decreasing oxidative stress and inflammation and the dosage of 10mg/kg could be apparently better than 20mg/kg. Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.

  10. The effects of sildenafil in liver and kidney injury in a rat model of severe scald burn: a biochemical and histopathological study.

    Science.gov (United States)

    Gökakın, Ali Kağan; Atabey, Mustafa; Deveci, Koksal; Sancakdar, Enver; Tuzcu, Mehmet; Duger, Cevdet; Topcu, Omer

    2014-09-01

    Severe burn induces systemic inflammation and reactive oxygen species leading to lipid peroxidation which may play role in remote organs injury. Sildenafil is a selective and potent inhibitor of cyclic guanosine monophosphate specific phosphodiesterase-5. Sildenafil reduces oxidative stress and inflammation in distant organs. The aim of the present study was to evaluate the effects of different dosages of sildenafil in remote organs injury. A total of thirty-two rats were randomly divided into four equal groups. The groups were designated as follows: Sham, Control, 10, and T20 mg/kg sildenafil treatment groups. Levels of malondialdehyde (MDA), vascular endothelial growth factor (VEGF), VEGF receptor (Flt-1), activities of glutathione peroxidase (Gpx), levels of total antioxidative capacity (TAC), and total oxidant status (TOS) were measured in both tissues and serum, and a semi-quantitative scoring system was used for the evaluation of histopathological findings. Sildenafil increased levels of Gpx, and Flt-1, and decreased MDA and VEGF levels in tissues. Sildenafil also increased serum levels of TAC and Flt-1 and decreased TOS, OSI, and VEGF. Sildenafil decreased inflammation scores in remote organs in histopathological evaluation. It has protective effects in severe burn-related remote organ injuries by decreasing oxidative stress and inflammation.

  11. Beneficial effects of combined benazepril-amlodipine on cardiac nitric oxide, cGMP, and TNF-alpha production after cardiac ischemia.

    Science.gov (United States)

    Siragy, Helmy M; Xue, Chun; Webb, Randy L

    2006-05-01

    The aim of this study was to determine if myocardial inflammation is increased after myocardial ischemia and whether angiotensin-converting enzyme inhibitors, calcium channel blockers, or diuretics decrease mediators of inflammation in rats with induced myocardial ischemia. Changes in cardiac interstitial fluid (CIF) levels of nitric oxide metabolites (NOX), cyclic guanosine 3',5'-monophosphate (cGMP), angiotensin II (Ang II), and tumor necrosis factor-alpha (TNF-alpha) were monitored with/without oral administration of benazepril, amlodipine, combined benazepril-amlodipine, or hydrochlorothiazide. Using a microdialysis technique, levels of several mediators of inflammation were measured after sham operation or 30-minute occlusion of the left anterior descending coronary artery. Compared with sham animals, levels of CIF NOX and cGMP were decreased in animals with ischemia (P Benazepril or amlodipine significantly increased NOX levels (P benazepril significantly increased cGMP (P benazepril-amlodipine further increased CIF NOX and cGMP (P benazepril alone, or combined benazepril-amlodipine significantly reduced TNF-alpha (P benazepril-amlodipine may be beneficial for managing cardiac ischemia.

  12. Analysis of proton wires in the enzyme active site suggests a mechanism of c-di-GMP hydrolysis by the EAL domain phosphodiesterases.

    Science.gov (United States)

    Grigorenko, Bella L; Knyazeva, Marina A; Nemukhin, Alexander V

    2016-11-01

    We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Ginger Extract Inhibits Biofilm Formation by Pseudomonas aeruginosa PA14

    Science.gov (United States)

    Kim, Han-Shin; Park, Hee-Deung

    2013-01-01

    Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger’s ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39–56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

  14. Plant natriuretic peptides: Systemic regulators of plant homeostasis and defense that can affect cardiomyoblasts

    KAUST Repository

    Gehring, Christoph A; Irving, Helen R.

    2010-01-01

    Immunologic evidence has suggested the presence of biologically active natriuretic peptide (NPs) hormones in plants because antiatrial NP antibodies affinity purify biologically active plant NPs (PNP). In the model plant, an Arabidopsis thaliana PNP (AtPNP-A) has been identified and characterized. AtPNP-A belongs to a novel class of molecules that share some similarity with the cell wall loosening expansins but do not contain the carbohydrate-binding wall anchor thus suggesting that PNPs and atrial natriuretic peptides are heterologs. AtPNP-A acts systemically, and this is consistent with its localization in the apoplastic extracellular space and the conductive tissue. Furthermore, AtPNP-A signals via the second messenger cyclic guanosine 3′,5′-monophosphate and modulates ion and water transport and homeostasis. It also plays a critical role in host defense against pathogens. AtPNP-A can be classified as novel paracrine plant hormone because it is secreted into the apoplastic space in response to stress and can enhance its own expression. Interestingly, purified recombinant PNP induces apo-ptosis in a dose-dependent manner and was most effective on cardiac myoblast cell lines. Because PNP is mimicking the effect of ANP in some instances, PNP may prove to provide useful leads for development of novel therapeutic NPs. Copyright © 2013 by The American Federation for Medical Research.

  15. Gene adaptation to extreme environments

    International Nuclear Information System (INIS)

    Marlaire, P.; Rodriguez, V.; Kerner, N.

    2005-01-01

    Full text: This work is oriented to the study of gene adaptation to extreme conditions, such as the hydrothermal system located in Copahue, Neuquen, Argentina. The organisms living there develop under two pressure selection conditions: the high temperature of thermal water and the strong impact of ultraviolet (UV) radiation. Several microorganisms found in this region were isolated and different colonies resistant to UV radiation were selected, a Geobacillus thermoleovorans strain identified through 16S RNA sequence, being the most remarkable. A gene library was prepared out of this strain with UV sensitive bacteria BH200 (uvrA::Tn10). A number of clones were isolated by means of UV selection, the most outstanding being a gene carrier able to codify for the guanosine monophosphate synthetase enzyme (GMPs). The suitability of said enzyme was proved by means of additional assays performed on ght 1 bacteria (guaA26::Tn 10) which lacked the enzyme. A transcript of 1100 pb was detected through Northern Blot. The result was consistent with that obtained for the mapping of the starting transcription site. The cloned GMPs produces an increase in growth speed and a greater biomass in BH200 bacteria. (author)

  16. Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid-rafts and its internalization in human endometrial cells.

    Science.gov (United States)

    Banadakoppa, Manu; Goluszko, Pawel; Liebenthal, Daniel; Yallampalli, Chandra

    2012-10-01

    Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the G(M1)-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.

  17. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    Energy Technology Data Exchange (ETDEWEB)

    Carvalho-Costa, P.G. [Programa de Graduação em Psicobiologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Branco, L.G.S. [Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Leite-Panissi, C.R.A. [Programa de Graduação em Psicobiologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-09-19

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

  18. Intraerythrocytic organic phosphates and hemoglobins of skua - Catharacta maccormicki (Stercoraridae: at two different stages of the year in relation to Antartic migration

    Directory of Open Access Journals (Sweden)

    Gustavo Fraga Landini

    2013-08-01

    Full Text Available Catharacta maccormicki blood samples were collected in the winter (October and in the summer (February in order to study the intraerythrocytic organic phosphates, hemoglobin (Hb electrophoretic patterns, oxygen blood equilibrium and stripped Hbs, as well as the effect of 2,3-biphosphoglycerate (BPG and inositol hexaphosphate (IHP on oxygen affinity. All the samples (five from the winter and five from the summer showed the same electrophoretic pattern: one minor fast component and one major slow one. No differences in oxygen affinity and Bohr effect in the samples collected in the winter and in the summer were found. Oxygen affinity was higher in the stripped Hb than in the blood. BPG seemed to have no effect on the functional properties of skua Hb while IHP does. No BPG was found in any sample. Both inositol pentaphosphate (IP5 and IHP were found in all the samples. The IP5/IHP ratio in the winter samples was 3.0 while in summer 3.5. Adenosine diphosphate (ADP was found in samples from both the seasons. Adenosine monophosphate (AMP and adenosine triphosphate (ATP were present only in the summer samples while guanosine triphosphate (GTP was found in the winter samples. Since IP5 and IHP are very powerful HB allosteric effectors, ATP and GTP might function as other protein modulators.

  19. Food-Grade Synthesis of Maillard-Type Taste Enhancers Using Natural Deep Eutectic Solvents (NADES

    Directory of Open Access Journals (Sweden)

    Maximilian Kranz

    2018-01-01

    Full Text Available The increasing demand for healthier food products, with reduced levels of table salt, sugar, and mono sodium glutamate, reinforce the need for novel taste enhancers prepared by means of food-grade kitchen-type chemistry. Although several taste modulating compounds have been discovered in processed foods, their Maillard-type ex food production is usually not exploited by industrial process reactions as the yields of target compounds typically do not exceed 1–2%. Natural deep eutectic solvents (NADES are reported for the first time to significantly increase the yields of the taste enhancers 1-deoxy-ᴅ-fructosyl-N-β-alanyl-ʟ-histidine (49% yield, N-(1-methyl-4-oxoimidazolidin-2-ylidene aminopropionic acid (54% yield and N2-(1-carboxyethyl guanosine 5′-monophosphate (22% yield at low temperature (80–100 °C within a maximum reaction time of 2 h. Therefore, NADES open new avenues to a “next-generation culinary chemistry” overcoming the yield limitations of traditional Maillard chemistry approaches and enable a food-grade Maillard-type generation of flavor modulators.

  20. Evaluation of Antinociceptive Activity of Ethanol Extract of Leaves of Adenanthera pavonina

    Directory of Open Access Journals (Sweden)

    Md. Moniruzzaman

    2015-01-01

    Full Text Available Adenanthera pavonina is a deciduous tree commonly used in the traditional medicine to treat inflammation and rheumatism. The aim of this study was to evaluate the antinociceptive activity of ethanol extract of leaves of A. pavonina (EEAP. EEAP was investigated using various nociceptive models induced thermally or chemically in mice including hot plate and tail immersion test, acetic acid-induced writhing, and glutamate- and formalin-induced licking tests at the doses of 50, 100, and 200 mg/kg body weight (p.o.. In addition, to assess the possible mechanisms, involvement of opioid system was verified using naloxone (2 mg/kg and cyclic guanosine monophosphate (cGMP signaling pathway by methylene blue (MB; 20 mg/kg. The results have demonstrated that EEAP produced a significant and dose-dependent increment in the hot plate latency and tail withdrawal time. It also reduced the number of abdominal constrictions and paw lickings induced by acetic acid and glutamate respectively. EEAP inhibited the nociceptive responses in both phases of formalin test. Besides, the reversal effects of naloxone indicated the association of opioid receptors on the exertion of EEAP action centrally. Moreover, the enhancement of writhing inhibitory activity by MB suggests the possible involvement of cGMP pathway in EEAP-mediated antinociception. These results prove the antinociceptive activity of the leaves of A. pavonina and support the traditional use of this plant.

  1. Chemistry of the 8-Nitroguanine DNA Lesion: Reactivity, Labelling and Repair.

    Science.gov (United States)

    Alexander, Katie J; McConville, Matthew; Williams, Kathryn R; Luzyanin, Konstantin V; O'Neil, Ian A; Cosstick, Richard

    2018-02-26

    The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 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...

  3. The transcription factor cyclic adenosine 3',5'-monophosphate response element-binding protein enhances the odonto/osteogenic differentiation of stem cells from the apical papilla.

    Science.gov (United States)

    Su, S; Zhu, Y; Li, S; Liang, Y; Zhang, J

    2017-09-01

    To investigate the role of cAMP response element-binding protein (CREB) in the regulation of odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs). Stem cells from the apical papilla were obtained from human impacted third molars (n = 15). Isolated SCAPs were transfected with CREB overexpressing/silenced lentivirus. Transfected cells were stained with alizarin red to investigate mineralized nodule formation. The expression of the mineralization-related genes, alkaline phosphatase (ALP), collagen type I (Col I), runt-related transcription factor 2 (RUNX2), osterix (OSX) and osteocalcin (OCN), was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Protein expression of the odontogenic-related marker dentine sialoprotein (DSP) and the osteogenic-related marker RUNX2 was measured by Western blotting analysis. One-way analysis of variance (anova) and Student's t-test were used for statistical analysis (a = 0.05). The overexpression of CREB enhanced mineralized nodule formation and up-regulated (P odonto/osteogenic-related markers, including ALP, Col I, RUNX2, OSX and OCN, and also increased (P odonto/osteogenic-related markers. Up-regulation of CREB expression promoted odonto/osteogenic differentiation of SCAPs and provided a potential method for the regeneration of the dentine-pulp complex. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  4. Thyroid hormone activates rat liver adenosine 5,-monophosphate-activated protein kinase: relation to CaMKKb, TAK1 and LKB1 expression and energy status.

    Science.gov (United States)

    Vargas, R; Ortega, Y; Bozo, V; Andrade, M; Minuzzi, G; Cornejo, P; Fernandez, V; Videla, L A

    2013-01-01

    AMP-activated protein kinase (AMPK) is a sensor of energy status supporting cellular energy homeostasis that may represent the metabolic basis for 3,3,,5-triiodo-L-thyronine (T3) liver preconditioning. Functionally transient hyperthyroid state induced by T3 (single dose of 0.1 mg/kg) in fed rats led to upregulation of mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of hepatic AMPK at 8 to 36 h after treatment. AMPK Thr 172 phosphorylation induced by T3 is associated with enhanced mRNA expression of the upstream kinases Ca2+ -calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) and transforming growth-factor-beta-activated kinase-1 (TAK1), with increased protein levels of CaMKKbeta and higher TAK1 phosphorylation, without changes in those of the liver kinase B1 (LKB1) signaling pathway. Liver contents of AMP and ADP were augmented by 291 percent and 44 percent by T3 compared to control values (p less than 0.05), respectively, whereas those of ATP decreased by 64% (p less than 0.05), with no significant changes in the total content of adenine nucleotides (AMP + ADP + ATP) at 24 h after T3 administration. Consequently, hepatic ATP/ADP content ratios exhibited 64 percent diminution (p less than 0.05) and those of AMP/ATP increased by 425 percent (p less than 0.05) in T3-treated rats over controls. It is concluded that in vivoT3 administration triggers liver AMPK upregulation in association with significant enhancements in AMPK mRNA expression, AMPK phosphorylation coupled to CaMKKbeta and TAK1 activation, and in AMP/ATP ratios, which may promote enhanced AMPK activity to support T3-induced energy consuming processes such as those of liver preconditioning.

  5. A biphasic and brain-region selective down-regulation of cyclic adenosine monophosphate concentrations supports object recognition in the rat.

    Directory of Open Access Journals (Sweden)

    Maïte Hotte

    Full Text Available BACKGROUND: We aimed to further understand the relationship between cAMP concentration and mnesic performance. METHODS AND FINDINGS: Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p., rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p. and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p. before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold, prefrontal (1.3 fold and perirhinal (1.3 fold cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold from drug-treated rats (except for milrinone+RS 67333 treatment. After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold and the perirhinal cortex (2.1 fold from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold and milrinone (respectively 1.46 fold and 1.56 fold-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h. CONCLUSIONS: Our results strongly suggest that a "pre-sample" early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay.

  6. 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.

  7. Glycolytic pathway (GP), kreb's cycle (KC), and hexose monophosphate shunt (HMS) activity in myocardial subcellular fractions exposed to cannabinoids

    Energy Technology Data Exchange (ETDEWEB)

    Watson, A.T.; Manno, B.R.; King, J.W.; Fowler, M.R.; Dempsey, C.A.; Manno, J.E.

    1986-03-05

    Delta-9-tetrahydrocannabinol (..delta../sup 9/-THC), the primary psychoactive component of marihuana, and its active metabolite 11-hydroxy-..delta../sup 9/-tetrahydrocannabinol (11-OH-..delta../sup 9/-THC) have been reported to produce a direct cardiac depressant effect. Studies in isolated perfused rat hearts have indicated a decreased force of contraction (inotropic response) when ..delta../sup 9/-THC or 11-OH-..delta../sup 9/-THC was administered in microgram amounts. The mechanism and site of action have not been explained or correlated with associated metabolic pathways. The purpose of this study was to investigate the effects of cannabinoids on major myocardial energy producing pathways, GP and KC, and a non-energy producing pathway, HMS. Cardiac ventricular tissue from male Sprague-Dawley rats (250-300 g) was excised and homogenized for subcellular fractionation. KC, GP and HMS activity was assayed in the appropriate fractions by measuring /sup 14/CO/sub 2/ generation from /sup 14/C-2-pyruvate, /sup 14/C-6-glucose and /sup 14/C-1-glucose respectively. Duplicate assays (n=8) were performed on tissue exposed to saline (control), empty liposomes (vehicle) and four doses each of ..delta../sup 9/-THC and 11-OH-..delta../sup 9/-THC. Changes in metabolic activity and decreases in cardiac contractile performance may be associated.

  8. Inhibition of Cyclic Adenosine Monophosphate (cAMP-response Element-binding Protein (CREB-binding Protein (CBP/β-Catenin Reduces Liver Fibrosis in Mice

    Directory of Open Access Journals (Sweden)

    Yosuke Osawa

    2015-11-01

    Full Text Available Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4- or bile duct ligation (BDL-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis.

  9. [Accumulation of cyclic adenosine monophosphate in the ovary of the eel (Anguilla anguilla L.) in vitro under the effect of carp gonadotropin or ovine lutropin: kinetics and thermodependence].

    Science.gov (United States)

    Salmon, C; Marchelidon, J; Fontaine-Bertrand, E; Fontaine, Y A

    1986-01-01

    Cyclic AMP (cAMP) in pieces of eel ovary was greatly increased in vitro by the gonadotropin (cGTH) of carp, another teleost fish; after one hour at 20 degrees C, maximal stimulation = 31.7 and E.D. 50 = 0.08 micrograms/ml. Ovine lutropin (oLH) had less effect (maximal stimulation: 2.35; E.D. 50: 1.42 micrograms/ml); its action suggested that it involved a subfraction (oLH/cGTH RAc) of the receptor-adenylate cyclase (RAc) systems which mediate the action of cGTH. Another difference was the percentage of total cAMP accumulated under hormonal stimulation and released into the incubation medium; this percentage was much higher with oLH than with cGTH (47 vs 8% after one hour at 20 degrees C). This result might be explained by a localization of oLH/cGTH RAc in cells (theca ?) situated on the outside of the follicles and/or by a relative lack of cAMP binding proteins in the case of cAMP produced by oLH/cGTH RAc. Kinetic and thermodependence studies also disclosed hormone-dependent differences; at 5 degrees C, cAMP concentration was maximal after 40 min with oLH, whereas it was still increasing after 3 h with cGTH. Differences in the properties of phosphodiesterases and/or in the clearance rate of hormone-receptor (HR) complexes could explain these results. The set of RAc systems in eel ovary recognizing fish gonadotropin would then be heterogeneous; some of them would be endowed with original properties concerning receptor specificity and cAMP diffusion as well as associated phosphodiesterase activity and/or HR metabolism. We suggest that at a stage of evolution when a single sensu stricto GTH is present (instead of two in tetrapods), "isoreceptors", differing in specificity and in their fate after hormone binding, could be an important element in the fine regulation of gonadal functions.

  10. 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. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

    International Nuclear Information System (INIS)

    Ishikawa, S.; Okada, K.; Saito, T.

    1988-01-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ([Ca2+]i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased [Ca2+] significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of [Ca2+]i depended on the intracellular Ca pool, since an AVP-induced rise in [Ca2+]i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased 45 Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells

  12. ORF Alignment: NC_004431 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available EDL933] pdb|1KDT|B Chain B, ... Cytidine Monophosphate Kinase From E.Coli In Complex ... With... 2',3'-Dideoxy-Cytidine Monophosphate pdb|1KDT|A ... Chain A, Cytidine Monophosphate Kinase From E.Coli...|1KDR|B Chain B, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara-Cytidine Monophosp...hate ... pdb|1KDR|A Chain A, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara

  13. ORF Alignment: NC_002655 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available EDL933] pdb|1KDT|B Chain B, ... Cytidine Monophosphate Kinase From E.Coli In Complex ... With... 2',3'-Dideoxy-Cytidine Monophosphate pdb|1KDT|A ... Chain A, Cytidine Monophosphate Kinase From E.Coli...|1KDR|B Chain B, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara-Cytidine Monophosp...hate ... pdb|1KDR|A Chain A, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara

  14. ORF Alignment: NC_002695 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available EDL933] pdb|1KDT|B Chain B, ... Cytidine Monophosphate Kinase From E.Coli In Complex ... With... 2',3'-Dideoxy-Cytidine Monophosphate pdb|1KDT|A ... Chain A, Cytidine Monophosphate Kinase From E.Coli...|1KDR|B Chain B, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara-Cytidine Monophosp...hate ... pdb|1KDR|A Chain A, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara

  15. ORF Alignment: NC_000913 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available EDL933] pdb|1KDT|B Chain B, ... Cytidine Monophosphate Kinase From E.Coli In Complex ... With... 2',3'-Dideoxy-Cytidine Monophosphate pdb|1KDT|A ... Chain A, Cytidine Monophosphate Kinase From E.Coli...|1KDR|B Chain B, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara-Cytidine Monophosp...hate ... pdb|1KDR|A Chain A, Cytidine Monophosphate Kinase From ... E.Coli In Complex With Ara

  16. Examining a role for PKG Iα oxidation in the pathogenesis of cardiovascular dysfunction during diet-induced obesity.

    Science.gov (United States)

    Rudyk, Olena; Eaton, Philip

    2017-09-01

    Protein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state. Diet-induced obesity is associated with NOS uncoupling, which may in part contribute to the associated cardiovascular dysfunction due to exacerbated PKG Iα disulfide oxidation to the disulfide state. This is a rational hypothesis because PKG Iα oxidation is known to significantly contribute to heart failure that arises from chronic myocardial oxidative stress. Bovine arterial endothelial cells (BAECs) or smooth muscle cells (SMCs) were exposed to drugs that uncouple NOS. These included 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) which promotes its S-glutathiolation, 4-diamino-6-hydroxy-pyrimidine (DAHP) which inhibits guanosine-5'-triphosphate-cyclohydrolase 2 to prevent BH 4 synthesis or methotrexate (MTX) which inhibits the regeneration of BH 4 from BH 2 by dihydrofolate reductase. While all the drugs mentioned above induced robust PKG Iα disulfide dimerization in cells, exposure of BAECs to NOS inhibitor L-NMMA did not. Increased PKG Iα disulfide formation occurred in hearts and aortae from mice treated in vivo with DAHP (10mM in a drinking water for 3 weeks). Redox-dead C42S PKG Iα knock-in (KI) mice developed less pronounced cardiac posterior wall hypertrophy and did not develop cardiac dysfunction, assessed by echocardiography, compared to the wild-type (WT) mice after chronic DAHP treatment. WT or

  17. Protein (Cyanobacteria): 652996914 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available mine monophosphate kinase Planktothrix agardhii MLIKDIGEQGLLEIVKGFCPSEIVGDDAAILAVSGDESLVITTDMLVDEVHFSDRTTSPF...DVGWRGAAVNLSDLAAMGAFPIGITVALGITDNKTVSWVEQLYQGLTTCLNQYQTPIVGGDICRSAVTCISITAFGRVNPKLAIRRSVARPGDKIIVTGDHGDSRAGL

  18. The Arabidopsis thaliana proteome harbors undiscovered multi-domain molecules with functional guanylyl cyclase catalytic centers

    KAUST Repository

    Wong, Aloysius Tze

    2013-07-08

    Background: Second messengers link external cues to complex physiological responses. One such messenger, 3\\',5\\'-cyclic guanosine monophosphate (cGMP), has been shown to play a key role in many physiological responses in plants. However, in higher plants, guanylyl cyclases (GCs), enzymes that generate cGMP from guanosine-5\\'-triphosphate (GTP) have remained elusive until recently. GC search motifs constructed from the alignment of known GCs catalytic centers form vertebrates and lower eukaryotes have led to the identification of a number of plant GCs that have been characterized in vitro and in vivo.Presentation of the hypothesis.Recently characterized GCs in Arabidopsis thaliana contributed to the development of search parameters that can identify novel candidate GCs in plants. We hypothesize that there are still a substantial number (> 40) of multi-domain molecules with potentially functional GC catalytic centers in plants that remain to be discovered and characterized. Testing the hypothesis. The hypothesis can be tested, firstly, by computational methods constructing 3D models of selected GC candidates using available crystal structures as templates. Homology modeling must include substrate docking that can provide support for the structural feasibility of the GC catalytic centers in those candidates. Secondly, recombinant peptides containing the GC domain need to be tested in in vitro GC assays such as the enzyme-linked immune-sorbent assay (ELISA) and/or in mass spectrometry based cGMP assays. In addition, quantification of in vivo cGMP transients with fluorescent cGMP-reporter assays in wild-type or selected mutants will help to elucidate the biological role of novel GCs.Implications of the hypothesis.If it turns out that plants do harbor a large number of functional GC domains as part of multi-domain enzymes, then major new insights will be gained into the complex signal transduction pathways that link cGMP to fundamental processes such as ion transport

  19. 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

  20. Dynamic Changes of Endothelium Derived Factors and Cyclic Nucleotides in Ascites Broilers and Control of L-arginine

    Institute of Scientific and Technical Information of China (English)

    HAN Bo; WANG Xiao-long

    2003-01-01

    A flock of AA breed chickens were reared in peterstme brood-vait chamber using high energypelleted feed, at 14 days of age, 400 birds were separated into 3 groups randomly as follows: 100 birds wereexposed to normal ambient temperature (20℃) as control group, 150 birds were exposed to low ambient tem-perature (11℃) in order to induce ascites as treatment Ⅰ group, another 100 birds were also exposed to lowambient temperature (11℃) and fed the diets containing 1% L-arginine for ascitic prophylactic treatment astreatment Ⅱ group. The blood samples were collected on 3, 4, 5, 6, 7 wk, respectively, to measure the con-tents of plasma endothelin (ET-1), angiotensin Ⅱ (Ang Ⅱ ) and cyclic adenosine monophosphate (cAMP) andcyclic guanosine monophosphate (cGMP). The results indicated that the contents of cAMP, cGMP, Ang Ⅱand the ratio of cAMP/cGMP in treatment Ⅰ and ascitic broilers were higher than that of correspondent con-trol group(P<0.01, P<0.05), ET-1 of preascitic broilers were higher than that of control group(P<0.05),while there were insignificant differences with later ascitic broilers, the contents of cAMP and cGMP in treat-ment Ⅱ were higher than treatment Ⅰ and control group(P<0.01, P<0.05), whereas, the ratio of cAMP/cGMP and the contents of Ang Ⅱ were gradually decreased than that of control group(P<0.05), the contentsof ET-1 were not changed. By further analysis, the increased plasma Ang Ⅱ at low ambient temperature condi-tion in broilers made endothelium cell secretion of increased ET-1, cAMP, cGMP and decreased NO. So lowtemperature accelarated ascites syndrome in broilers. Supplemented L-arginine can decrease ET-1, cAMP andcAMP/cGMP. It is concluded that cAMP mediated pulmonary hypertension syndrome in broilers.

  1. Effects and Mechanism of Action of a Tribulus terrestris Extract on Penile Erection

    Science.gov (United States)

    Do, Jungmo; Choi, Seemin; Choi, Jaehwi

    2013-01-01

    Purpose Tribulus terrestris has been used as an aphrodisiac. However, little is known about the effects and mechanism of action of T. terrestris on penile erection. Therefore, the effect of a T. terrestris extract and the mechanism of action of the extract on relaxation of the corpus cavernosum (CC) were investigated. The erectogenic effects of an oral preparation of the extract were also assessed. Materials and Methods The relaxation effects and mechanism of action of the T. terrestris extract on rabbit CC were investigated in an organ bath. The intracavernous pressure (ICP) was calculated after oral administration of the extract for 1 month to evaluate whether the relaxation response of the CC shown in the organ bath occurred in vivo. Additionally, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in the CC by immunoassay. Smooth muscle relaxation was expressed as the percentage decrease in precontraction induced by phenylephrine. The ICP was also assessed in rats after oral administration of the extract for 1 month, and changes in concentrations of cGMP and cAMP were monitored. Results Concentration-dependent relaxation effects of the extract on the CC were detected in the organ bath study. Relaxation of the CC by the T. terrestris extract was inhibited in both an endothelium-removed group and an L-arginen methyl ester pretreatment group. The ICP measured after oral administration of the T. terrestris extract for 1 month was higher than that measured in the control group, and a significant increase in cAMP was observed in the T. terrestris extract group. Conclusions The T. terrestris extract induced concentration-dependent relaxation of the CC in an organ bath. The mechanism included a reaction involving the nitric oxide/nitric oxide synthase pathway and endothelium of the CC. Moreover, in an in vivo study, the T. terrestris extract showed a significant concentration-dependent increase in ICP. Accordingly, the T

  2. Cyclic GMP-mediated memory enhancement in the object recognition test by inhibitors of phosphodiesterase-2 in mice.

    Science.gov (United States)

    Lueptow, Lindsay M; Zhan, Chang-Guo; O'Donnell, James M

    2016-02-01

    Cyclic nucleotide phosphodiesterase-2 (PDE2) is a potential therapeutic target for the treatment of cognitive dysfunction. Using the object recognition test (ORT), this study assessed the effects of two PDE2 inhibitors, Bay 60-7550 and ND7001, on learning and memory, and examined underlying mechanisms. To assess the role of PDE2 inhibition on phases of memory, Bay 60-7550 (3 mg/kg) was administered: 30 min prior to training; 0, 1, or 3 h after training; or 30 min prior to recall testing. To assess cyclic nucleotide involvement in PDE2 inhibitor-enhanced memory consolidation, either the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg; intraperitoneal (IP)), soluble guanylyl cyclase inhibitor 1H-[-1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 20 mg/kg; IP), protein kinase G inhibitor KT5823 (2.5 μg; intracerebroventricular (ICV)), or protein kinase A inhibitor H89 (1 μg; ICV) was administered 30 min prior to the PDE2 inhibitor Bay 60-7550 (3 mg/kg) or ND7001 (3 mg/kg). Changes in the phosphorylation of 3'5'-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) at Ser-133 and vasodilator-stimulated phosphoprotein (VASP) at Ser-239 were determined to confirm activation of cAMP and 3'5'-cyclic guanosine monophosphate (cGMP) signaling. Bay 60-7550 (3 mg/kg) enhanced memory of mice in the ORT when given 30 min prior to training, immediately after training, or 30 min prior to recall. Inhibitors of the cGMP pathway blocked the memory-enhancing effects of both Bay 60-7550 (3 mg/kg) and ND7001 (3 mg/kg) on early consolidation processes. Bay 60-7550 (3 mg/kg) enhanced phosphorylation of CREB and VASP, both targets of cGMP-dependent protein kinase (PKG). These results confirm a potential of PDE2, or components of its signaling pathway, as a therapeutic target for drug discovery focused on restoring memory function.

  3. Effects of Biotin Supplementation in the Diet on Adipose Tissue cGMP Concentrations, AMPK Activation, Lipolysis, and Serum-Free Fatty Acid Levels.

    Science.gov (United States)

    Boone-Villa, Daniel; Aguilera-Méndez, Asdrubal; Miranda-Cervantes, Adriana; Fernandez-Mejia, Cristina

    2015-10-01

    Several studies have shown that pharmacological concentrations of biotin decrease hyperlipidemia. The molecular mechanisms by which pharmacological concentrations of biotin modify lipid metabolism are largely unknown. Adipose tissue plays a central role in lipid homeostasis. In the present study, we analyzed the effects of biotin supplementation in adipose tissue on signaling pathways and critical proteins that regulate lipid metabolism, as well as on lipolysis. In addition, we assessed serum fatty acid concentrations. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (control: 1.76 mg biotin/kg; supplemented: 97.7 mg biotin/kg diet) over 8 weeks postweaning. Compared with the control group, biotin-supplemented mice showed an increase in the levels of adipose guanosine 3',5'-cyclic monophosphate (cGMP) (control: 30.3±3.27 pmol/g wet tissue; supplemented: 49.5±3.44 pmol/g wet tissue) and of phosphorylated forms of adenosine 5'-monophosphate-activated protein kinase (AMPK; 65.2%±1.06%), acetyl-coenzyme A (CoA), carboxylase-1 (196%±68%), and acetyl-CoA carboxylase-2 (78.1%±18%). Serum fatty acid concentrations were decreased (control: 1.12±0.04 mM; supplemented: 0.91±0.03 mM), and no change in lipolysis was found (control: 0.29±0.05 μmol/mL; supplemented: 0.33±0.08 μmol/mL). In conclusion, 8 weeks of dietary biotin supplementation increased adipose tissue cGMP content and protein expression of the active form of AMPK and of the inactive forms of acetyl-CoA carboxylase-1 and acetyl-CoA carboxylase-2. Serum fatty acid levels fell, and no change in lipolysis was observed. These findings provide insight into the effects of biotin supplementation on adipose tissue and support its use in the treatment of dyslipidemia.

  4. Orexin-A potentiates L-type calcium/barium currents in rat retinal ganglion cells.

    Science.gov (United States)

    Liu, F; Weng, S-J; Yang, X-L; Zhong, Y-M

    2015-10-01

    Two neuropeptides, orexin-A and orexin-B (also called hypocretin-1 and -2), have been implicated in sleep/wake regulation, feeding behaviors via the activation of two subtypes of G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). While the expression of orexins and orexin receptors is immunohistochemically revealed in retinal neurons, the function of these peptides in the retina is largely unknown. Using whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that orexin-A increased L-type-like barium currents (IBa,L) in ganglion cells (GCs), and the effect was blocked by the selective OX1R antagonist SB334867, but not by the OX2R antagonist TCS OX2 29. The orexin-A effect was abolished by intracellular dialysis of GDP-β-S/GPAnt-2A, a Gq protein inhibitor, suggesting the mediation of Gq. Additionally, during internal dialysis of the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor U73122, orexin-A did not change the IBa,L of GCs, whereas the orexin-A effect persisted in the presence of the phosphatidylcholine (PC)-PLC inhibitor D609. The orexin-A-induced potentiation was not seen with internal infusion of Ca(2+)-free solution or when inositol 1,4,5-trisphosphate (IP3)-sensitive Ca(2+) release from intracellular stores was blocked by heparin/xestospongins-C. Moreover, the orexin-A effect was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, but was eliminated when PKC was inhibited by bisindolylmaleimide IV (Bis-IV)/Gö6976. Neither adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) nor guanosine 3',5'-cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway was likely involved, as orexin-A persisted to potentiate the IBa,L of GCs no matter these two pathways were activated or inhibited. These results suggest that, by activating OX1R, orexin-A potentiates the IBa,L of rat GCs through a distinct Gq/PI-PLC/IP3/Ca(2+)/PKC signaling pathway. Copyright

  5. In vitro and in vivo investigation of natural compounds from seed extract of Mucuna pruriens lacking l-DOPA for the treatment of erectile dysfunction.

    Science.gov (United States)

    Duangnin, Natthachai; Phitak, Thanyaluck; Pothacharoen, Peraphan; Kongtawelert, Prachya

    2017-03-01

    To investigate the biological effects of the Mucuna pruriens (M. pruriens) seed extracts that lacked l-DOPA, which was formerly reported as the active ingredient, on erectile dysfunction (ED) both in vitro and in vivo. Seed of M. pruriens plant that cultivated in Mae Taeng District, Chiang Mai Province, Thailand, was collected. Component of its seeds were extracted and isolated into 2 fractions using methanol, polar and nonpolar. Each fraction was investigated for phytochemicals using gas chromatography and mass spectroscopy and was screened for biological activity in vitro using three different cell lines. The most biological active fraction was used to treat both streptozotocin (STZ)-induced diabetes mellitus-erectile dysfunction (DM-ED) male Wistar rats and normal rats (n = 6 per groups) to compare the effect on sexual behavior parameters, including number of intromission, mounting and ejaculation, with that of rats given Sildenafil by individually pairing with their female counterparts. Penile tissues and serums were collected to determine histological structure, related gene expression and biomolecules. The phytochemicals of the polar fraction were possibly catechol and its derivatives plus polyphenols, whereas the nonpolar fraction consisted of lipid derivatives. l-DOPA was not detected in either of the extracts. The polar fraction was able to up-regulate the expression of ED-related genes including eNOS and nNOS in vitro which subsequently promotes nitric oxide production and maintains intracellular cyclic guanosine monophosphate levels. When administrated to DM-ED rats, the polar extract significantly improved all sexual behavior parameters in DM-ED rats compared to untreated group (18.3 ± 1.8 to 10.8 ± 2.9 for intromission, 9.8 ± 2.2 to 5.7 ± 1.3 for mounting, and 1.8 ± 0.6 to 0.2 ± 0.4 for ejaculation). That effect might due to the ability of the extract to stimulate the expression of eNOS and nNOS which results in nitric oxide

  6. Impact of the NO-Sensitive Guanylyl Cyclase 1 and 2 on Renal Blood Flow and Systemic Blood Pressure in Mice.

    Science.gov (United States)

    Mergia, Evanthia; Thieme, Manuel; Hoch, Henning; Daniil, Georgios; Hering, Lydia; Yakoub, Mina; Scherbaum, Christina Rebecca; Rump, Lars Christian; Koesling, Doris; Stegbauer, Johannes

    2018-03-23

    Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S -nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.

  7. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

    Directory of Open Access Journals (Sweden)

    S.C.F. Olinto

    2012-11-01

    Full Text Available The amino acid arginine (Arg is a recognized secretagogue of growth hormone (GH, and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO, which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g were removed, divided into two halves, pooled (three hemi-pituitaries and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM, the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM and a cyclic guanosine monophosphate (cGMP analogue (8-Br-cGMP, 1 mM increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS inhibitor, 55 mM abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

  8. Systemic induction of NO-, redox- and cGMP signalling in the pumpkin extrafascicular phloem upon local leaf wounding

    Directory of Open Access Journals (Sweden)

    Frank eGaupels

    2016-02-01

    Full Text Available Cucurbits developed the unique extrafascicular phloem (EFP as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima. Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint towards a wound-induced shift in the redox status of the EFP. Nitric oxide (NO is another important player in stress-induced redox signalling in plants. Therefore, we analysed NO-dependent protein modifications in the EFP. Six to 48 h after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1 and Cyclophilin 18 (CYP18 as well as the 26.5 kD isoform of Phloem Protein 2 (PP2 were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP in the EFP and discuss the possible function of this second messenger in systemic NO and redox signalling within the EFP.

  9. Lebetin 2, a Snake Venom-Derived Natriuretic Peptide, Attenuates Acute Myocardial Ischemic Injury through the Modulation of Mitochondrial Permeability Transition Pore at the Time of Reperfusion.

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    Bochra Tourki

    Full Text Available Cardiac ischemia is one of the leading causes of death worldwide. It is now well established that natriuretic peptides can attenuate the development of irreversible ischemic injury during myocardial infarction. Lebetin 2 (L2 is a new discovered peptide isolated from Macrovipera lebetina venom with structural similarity to B-type natriuretic peptide (BNP. Our objectives were to define the acute cardioprotective actions of L2 in isolated Langendorff-perfused rat hearts after regional or global ischemia-reperfusion (IR. We studied infarct size, left ventricular contractile recovery, survival protein kinases and mitochondrial permeability transition pore (mPTP opening in injured myocardium. L2 dosage was determined by preliminary experiments at its ability to induce cyclic guanosine monophosphate (cGMP release without changing hemodynamic effects in normoxic hearts. L2 was found to be as effective as BNP in reducing infarct size after the induction of either regional or global IR. Both peptides equally improved contractile recovery after regional IR, but only L2 increased coronary flow and reduced severe contractile dysfunction after global ischemia. Cardioprotection afforded by L2 was abolished after isatin or 5-hydroxydecanote pretreatment suggesting the involvement of natriuretic peptide receptors and mitochondrial KATP (mitoKATP channels in the L2-induced effects. L2 also increased survival protein expression in the reperfused myocardium as evidenced by phosphorylation of signaling pathways PKCε/ERK/GSK3β and PI3K/Akt/eNOS. IR induced mitochondrial pore opening, but this effect was markedly prevented by L2 treatment. These data show that L2 has strong cardioprotective effect in acute ischemia through stimulation of natriuretic peptide receptors. These beneficial effects are mediated, at least in part, by mitoKATP channel opening and downstream activated survival kinases, thus delaying mPTP opening and improving IR-induced mitochondrial

  10. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Yang, Ying, E-mail: yangying_sh@yahoo.com [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Shen, Weili, E-mail: weili_shen@hotmail.com [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

  11. Nitric oxide signalling and neuronal nitric oxide synthase in the heart under stress.

    Science.gov (United States)

    Zhang, Yin Hua

    2017-01-01

    Nitric oxide (NO) is an imperative regulator of the cardiovascular system and is a critical mechanism in preventing the pathogenesis and progression of the diseased heart. The scenario of bioavailable NO in the myocardium is complex: 1) NO is derived from both endogenous NO synthases (endothelial, neuronal, and/or inducible NOSs [eNOS, nNOS, and/or iNOS]) and exogenous sources (entero-salivary NO pathway) and the amount of NO from exogenous sources varies significantly; 2) NOSs are located at discrete compartments of cardiac myocytes and are regulated by distinctive mechanisms under stress; 3) NO regulates diverse target proteins through different modes of post-transcriptional modification (soluble guanylate cyclase [sGC]/cyclic guanosine monophosphate [cGMP]/protein kinase G [PKG]-dependent phosphorylation, S -nitrosylation, and transnitrosylation); 4) the downstream effectors of NO are multidimensional and vary from ion channels in the plasma membrane to signalling proteins and enzymes in the mitochondria, cytosol, nucleus, and myofilament; 5) NOS produces several radicals in addition to NO (e.g. superoxide, hydrogen peroxide, peroxynitrite, and different NO-related derivatives) and triggers redox-dependent responses. However, nNOS inhibits cardiac oxidases to reduce the sources of oxidative stress in diseased hearts. Recent consensus indicates the importance of nNOS protein in cardiac protection under pathological stress. In addition, a dietary regime with high nitrate intake from fruit and vegetables together with unsaturated fatty acids is strongly associated with reduced cardiovascular events. Collectively, NO-dependent mechanisms in healthy and diseased hearts are better understood and shed light on the therapeutic prospects for NO and NOSs in clinical applications for fatal human heart diseases.

  12. From field to health: a simple way to increase the nutraceutical content of grape as shown by NO-dependent vascular relaxation.

    Science.gov (United States)

    Fumagalli, Francesca; Rossoni, Mara; Iriti, Marcello; di Gennaro, Antonio; Faoro, Franco; Borroni, Emanuele; Borgo, Michele; Scienza, Attilio; Sala, Angelo; Folco, Giancarlo

    2006-07-26

    Polyphenolic grapevine components involved in plant resistance against pathogens possess various pharmacological properties that include nitric oxide (NO)-dependent vasodilation and anti-inflammatory and free radical scavenging activities, which may explain the protective effect of moderate red wine consumption against cardiovascular disease. The aim of this work was (a) to verify the possibility that preharvest treatments of grapevine with a plant activator, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), could lead to an enriched nutraceutical potential of wine and (b) to characterize the profile of metabolites responsible for pharmacological activity. Plant spraying at the end of veraison, with a water suspension of BTH (0.3 mM), led to increased whole anthocyanin content as confirmed by HPLC comparative analysis. Extracts from berry skins of BTH-treated grapevines caused NO-dependent vasorelaxation, with a concentration-response curve that was significantly shifted to the left of the control non-BTH-treated curve. Moreover, 1:1000 dilutions of berry extracts from BTH-treated plants significantly increased basal production of guanosine 3',5'-cyclic monophosphate (cGMP) in human vascular endothelial cells when compared to the corresponding extracts of untreated plants. These results show that BTH treatment increases anthocyanin content of grape extracts, as well as their ability to induce NO-mediated vasoprotection. No increase of anthocyanin content was observed in the wine extracts from BTH-treated vines. It is concluded that BTH treatment could be exploited to increase the nutraceutical potential of grapes.

  13. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

    International Nuclear Information System (INIS)

    Forte, L.R.; Krause, W.J.; Freeman, R.H.

    1988-01-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3',5'-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with 125 I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments

  14. High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity.

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    Pfeilmeier, Sebastian; Saur, Isabel Marie-Luise; Rathjen, John Paul; Zipfel, Cyril; Malone, Jacob George

    2016-05-01

    The plant innate immune system employs plasma membrane-localized receptors that specifically perceive pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern-triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant-associated bacteria. Here, we show that cyclic-di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic-di-GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic-di-GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic-di-GMP signalling on bacterial behaviour. © 2015 THE AUTHORS MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

  15. Genetic heterogeneity and consanguinity lead to a "double hit": homozygous mutations of MYO7A and PDE6B in a patient with retinitis pigmentosa.

    Science.gov (United States)

    Goldenberg-Cohen, Nitza; Banin, Eyal; Zalzstein, Yael; Cohen, Ben; Rotenstreich, Ygal; Rizel, Leah; Basel-Vanagaite, Lina; Ben-Yosef, Tamar

    2013-01-01

    Retinitis pigmentosa (RP), the most genetically heterogeneous disorder in humans, actually represents a group of pigmentary retinopathies characterized by night blindness followed by visual-field loss. RP can appear as either syndromic or nonsyndromic. One of the most common forms of syndromic RP is Usher syndrome, characterized by the combination of RP, hearing loss, and vestibular dysfunction. The underlying cause of the appearance of syndromic and nonsyndromic RP in three siblings from a consanguineous Israeli Muslim Arab family was studied with whole-genome homozygosity mapping followed by whole exome sequencing. THE FAMILY WAS FOUND TO SEGREGATE NOVEL MUTATIONS OF TWO DIFFERENT GENES: myosin VIIA (MYO7A), which causes type 1 Usher syndrome, and phosphodiesterase 6B, cyclic guanosine monophosphate-specific, rod, beta (PDE6B), which causes nonsyndromic RP. One affected child was homozygous for both mutations. Since the retinal phenotype seen in this patient results from overlapping pathologies, one might expect to find severe retinal degeneration. Indeed, he was diagnosed with RP based on an abnormal electroretinogram (ERG) at a young age (9 months). However, this early diagnosis may be biased, as two of his older siblings had already been diagnosed, leading to increased awareness. At the age of 32 months, he had relatively good vision with normal visual fields. Further testing of visual function and structure at different ages in the three siblings is needed to determine whether the two RP-causing genes mutated in this youngest sibling confer increased disease severity. This report further supports the genetic heterogeneity of RP, and demonstrates how consanguinity could increase intrafamilial clustering of multiple hereditary diseases. Moreover, this report provides a unique opportunity to study the clinical implications of the coexistence of pathogenic mutations in two RP-causative genes in a human patient.

  16. Genetic heterogeneity and consanguinity lead to a “double hit”: Homozygous mutations of MYO7A and PDE6B in a patient with retinitis pigmentosa

    Science.gov (United States)

    Goldenberg-Cohen, Nitza; Banin, Eyal; Zalzstein, Yael; Cohen, Ben; Rotenstreich, Ygal; Rizel, Leah; Basel-Vanagaite, Lina

    2013-01-01

    Purpose Retinitis pigmentosa (RP), the most genetically heterogeneous disorder in humans, actually represents a group of pigmentary retinopathies characterized by night blindness followed by visual-field loss. RP can appear as either syndromic or nonsyndromic. One of the most common forms of syndromic RP is Usher syndrome, characterized by the combination of RP, hearing loss, and vestibular dysfunction. Methods The underlying cause of the appearance of syndromic and nonsyndromic RP in three siblings from a consanguineous Israeli Muslim Arab family was studied with whole-genome homozygosity mapping followed by whole exome sequencing. Results The family was found to segregate novel mutations of two different genes: myosin VIIA (MYO7A), which causes type 1 Usher syndrome, and phosphodiesterase 6B, cyclic guanosine monophosphate-specific, rod, beta (PDE6B), which causes nonsyndromic RP. One affected child was homozygous for both mutations. Since the retinal phenotype seen in this patient results from overlapping pathologies, one might expect to find severe retinal degeneration. Indeed, he was diagnosed with RP based on an abnormal electroretinogram (ERG) at a young age (9 months). However, this early diagnosis may be biased, as two of his older siblings had already been diagnosed, leading to increased awareness. At the age of 32 months, he had relatively good vision with normal visual fields. Further testing of visual function and structure at different ages in the three siblings is needed to determine whether the two RP-causing genes mutated in this youngest sibling confer increased disease severity. Conclusions This report further supports the genetic heterogeneity of RP, and demonstrates how consanguinity could increase intrafamilial clustering of multiple hereditary diseases. Moreover, this report provides a unique opportunity to study the clinical implications of the coexistence of pathogenic mutations in two RP-causative genes in a human patient. PMID:23882135

  17. Organic Nitrate Therapy, Nitrate Tolerance, and Nitrate-Induced Endothelial Dysfunction: Emphasis on Redox Biology and Oxidative Stress

    Science.gov (United States)

    2015-01-01

    Abstract Organic nitrates, such as nitroglycerin (GTN), isosorbide-5-mononitrate and isosorbide dinitrate, and pentaerithrityl tetranitrate (PETN), when given acutely, have potent vasodilator effects improving symptoms in patients with acute and chronic congestive heart failure, stable coronary artery disease, acute coronary syndromes, or arterial hypertension. The mechanisms underlying vasodilation include the release of •NO or a related compound in response to intracellular bioactivation (for GTN, the mitochondrial aldehyde dehydrogenase [ALDH-2]) and activation of the enzyme, soluble guanylyl cyclase. Increasing cyclic guanosine-3′,-5′-monophosphate (cGMP) levels lead to an activation of the cGMP-dependent kinase I, thereby causing the relaxation of the vascular smooth muscle by decreasing intracellular calcium concentrations. The hemodynamic and anti-ischemic effects of organic nitrates are rapidly lost upon long-term (low-dose) administration due to the rapid development of tolerance and endothelial dysfunction, which is in most cases linked to increased intracellular oxidative stress. Enzymatic sources of reactive oxygen species under nitrate therapy include mitochondria, NADPH oxidases, and an uncoupled •NO synthase. Acute high-dose challenges with organic nitrates cause a similar loss of potency (tachyphylaxis), but with distinct pathomechanism. The differences among organic nitrates are highlighted regarding their potency to induce oxidative stress and subsequent tolerance and endothelial dysfunction. We also address pleiotropic effects of organic nitrates, for example, their capacity to stimulate antioxidant pathways like those demonstrated for PETN, all of which may prevent adverse effects in response to long-term therapy. Based on these considerations, we will discuss and present some preclinical data on how the nitrate of the future should be designed. Antioxid. Redox Signal. 23, 899–942. PMID:26261901

  18. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    Science.gov (United States)

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  19. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  20. α2-Adrenoceptor Functionality in Postmortem Frontal Cortex of Depressed Suicide Victims

    Science.gov (United States)

    Valdizán, Elsa M.; Díez-Alarcia, Rebeca; González-Maeso, Javier; Pilar-Cuéllar, Fuencisla; García-Sevilla, Jesús A.; Meana, J. Javier; Pazos, Angel

    2013-01-01

    Background Alterations in brain density and signaling associated with monoamine receptors are believed to play a role in depressive disorders. This study evaluates the functional status of α2A-adrenoceptors in postmortem frontal cortex of depressed subjects. Methods G-protein activation and inhibition of adenylyl cyclase (AC) activity induced by the α2-adrenoceptor agonist UK14304 were measured in triplicate in samples from 15 suicide victims with an antemortem diagnosis of major depression and 15 matched control subjects. Results Basal [35S] guanosine γ thio-phosphate (GTPγS) binding and cyclic adenosine monophosphate accumulation did not differ between groups. In depressed victims, an increase in [35S] GTPγS binding potency (EC50 = .58 μmol/L vs. EC50 = 3.31 μmol/L; p < .01; depressed vs. control) and a significant reduction in the maximal inhibition of AC activity (Imax = 27 ± 4% vs. Imax = 47 ± 5%; p < .01) were observed after incubation with the α2-adrenoceptor agonist UK14304. No differences were found between antidepressant-free and antidepressant-treated subjects. A significant relationship between EC50 values for [35S] GTPγS and Imax values for AC assay was found (n = 30; r = −.43; p < .05). Conclusions The dual regulation of α2A-adrenoceptor signaling pathways raises the possibility that factors affecting the G-protein cycle and/or selective access of Gαi/o–protein to AC might be relevant to receptor abnormalities in depression, providing further support for the involvement of α2A-adrenoceptors in the pathogenesis of depression. PMID:20864091

  1. Heat shock factor-1 intertwines insulin/IGF-1, TGF-β and cGMP signaling to control development and aging

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    Barna János

    2012-11-01

    Full Text Available Abstract Background Temperature affects virtually all cellular processes. A quick increase in temperature challenges the cells to undergo a heat shock response to maintain cellular homeostasis. Heat shock factor-1 (HSF-1 functions as a major player in this response as it activates the transcription of genes coding for molecular chaperones (also called heat shock proteins that maintain structural integrity of proteins. However, the mechanisms by which HSF-1 adjusts fundamental cellular processes such as growth, proliferation, differentiation and aging to the ambient temperature remain largely unknown. Results We demonstrate here that in Caenorhabditis elegans HSF-1 represses the expression of daf-7 encoding a TGF-β (transforming growth factor-beta ligand, to induce young larvae to enter the dauer stage, a developmentally arrested, non-feeding, highly stress-resistant, long-lived larval form triggered by crowding and starvation. Under favorable conditions, HSF-1 is inhibited by crowding pheromone-sensitive guanylate cyclase/cGMP (cyclic guanosine monophosphate and systemic nutrient-sensing insulin/IGF-1 (insulin-like growth factor-1 signaling; loss of HSF-1 activity allows DAF-7 to promote reproductive growth. Thus, HSF-1 interconnects the insulin/IGF-1, TGF-β and cGMP neuroendocrine systems to control development and longevity in response to diverse environmental stimuli. Furthermore, HSF-1 upregulates another TGF-β pathway-interacting gene, daf-9/cytochrome P450, thereby fine-tuning the decision between normal growth and dauer formation. Conclusion Together, these results provide mechanistic insight into how temperature, nutrient availability and population density coordinately influence development, lifespan, behavior and stress response through HSF-1.

  2. The emperor's new clothes: PDE5 and the heart.

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    Chantal V Degen

    Full Text Available Phosphodiesterase-5 (PDE5 is highly expressed in the pulmonary vasculature, but its expression in the myocardium is controversial. Cyclic guanosine monophosphate (cGMP activates protein kinase G (PKG, which has been hypothesized to blunt cardiac hypertrophy and negative remodeling in heart failure. Although PDE5 has been suggested to play a significant role in the breakdown of cGMP in cardiomyocytes and hence PKG regulation in the myocardium, the RELAX trial, which tested effect of PDE5 inhibition on exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF failed to show a beneficial effect. These results highlight the controversy regarding the role and expression of PDE5 in the healthy and failing heart. This study used one- and two-dimensional electrophoresis and Western blotting to examine PDE5 expression in mouse (before and after trans-aortic constriction, dog (control and HFpEF as well as human (healthy and failing heart. We were unable to detect PDE5 in any cardiac tissue lysate, whereas PDE5 was present in the murine and bovine lung samples used as positive controls. These results indicate that if PDE5 is expressed in cardiac tissue, it is present in very low quantities, as PDE5 was not detected in either humans or any model of heart failure examined. Therefore in cardiac muscle, it is unlikely that PDE5 is involved the regulation of cGMP-PKG signaling, and hence PDE5 does not represent a suitable drug target for the treatment of cardiac hypertrophy. These results highlight the importance of rigorous investigation prior to clinical trial design.

  3. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

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    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  4. PKG in honey bees: spatial expression, Amfor gene expression, sucrose responsiveness, and division of labor.

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    Thamm, Markus; Scheiner, Ricarda

    2014-06-01

    Division of labor is a hallmark of social insects. In honey bees, division of labor involves transition of female workers from one task to the next. The most distinct tasks are nursing (providing food for the brood) and foraging (collecting pollen and nectar). The brain mechanisms regulating this form of behavioral plasticity have largely remained elusive. Recently, it was suggested that division of labor is based on nutrition-associated signaling pathways. One highly conserved gene associated with food-related behavior across species is the foraging gene, which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). Our analysis of this gene reveals the presence of alternative splicing in the honey bee. One isoform is expressed in the brain. Expression of this isoform is most pronounced in the mushroom bodies, the subesophageal ganglion, and the corpora allata. Division of labor and sucrose responsiveness in honey bees correlate significantly with foraging gene expression in distinct brain regions. Activating PKG selectively increases sucrose responsiveness in nurse bees to the level of foragers, whereas the same treatment does not affect responsiveness to light. These findings demonstrate a direct link between PKG signaling in distinct brain areas and division of labor. Furthermore, they demonstrate that the difference in sensory responsiveness between nurse bees and foragers can be compensated for by activating PKG. Our findings on the function of PKG in regulating specific sensory responsiveness and social organization offer valuable indications for the function of the cGMP/PKG pathway in many other insects and vertebrates. Copyright © 2013 Wiley Periodicals, Inc.

  5. Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.

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    Junbo Li

    Full Text Available Sodium nitrite, a common food additive, exists widely not only in the environment but also in our body. Excessive nitrite causes toxicological effects on human health; however, whether it affects vertebrate heart valve development remains unknown. In vertebrates, developmental defects of cardiac valves usually lead to congenital heart disease. To understand the toxic effects of nitrite on valvulogenesis, we exposed zebrafish embryos with different concentrations of sodium nitrite. Our results showed that sodium nitrite caused developmental defects of zebrafish heart dose dependently. It affected zebrafish heart development starting from 36 hpf (hour post fertilization when heart initiates looping process. Comprehensive analysis on the embryos at 24 hpf and 48 hpf showed that excessive nitrite did not affect blood circulation, vascular network, myocardium and endocardium development. But development of endocardial cells in atrioventricular canal (AVC of the embryos at 48 hpf was disrupted by too much nitrite, leading to defective formation of primitive valve leaflets at 76 hpf. Consistently, excessive nitrite diminished expressions of valve progenitor markers including bmp4, has2, vcana and notch1b at 48 hpf. Furthermore, 3', 5'-cyclic guanosine monophosphate (cGMP, downstream of nitric oxide (NO signaling, was increased its level significantly in the embryos exposed with excessive nitrite and microinjection of soluble guanylate cyclase inhibitor ODQ (1H-[1], [2], [4]Oxadiazolo[4,3-a] quinoxalin-1-one, an antagonist of NO signaling, into nitrite-exposed embryos could partly rescue the cardiac valve malformation. Taken together, our results show that excessive nitrite affects early valve leaflet formation by producing too much NO signaling.

  6. Pharmacokinetic, pharmacodynamic, and antihypertensive effects of the neprilysin inhibitor LCZ-696: sacubitril/valsartan.

    Science.gov (United States)

    Chrysant, Steven G

    2017-07-01

    LCZ-696, sacubitril/valsartan, is a dual-acting molecule consisting of the angiotensin II (Ang II) receptor blocker valsartan and the neprilysin (neutral endopeptidase) inhibitor AHU-377 with significant beneficial effects in patients with hypertension and heart failure (HF). Several recent studies have demonstrated a higher effectiveness of LCZ-696 compared to valsartan in the treatment of hypertension and HF. The rationale for the development and the Food and Drug Administration approval of LCZ-696 was based on the concept of an additive effect of the Ang II receptor blocker valsartan and the neutral endopeptidase (neprilysin) inhibitor AHU-377 for the treatment of hypertension and HF. The synergism from these drugs arises from the vasodilating effects of valsartan through its blockade of Ang II type 1 receptor and the action of natriuretic peptides atrial natriuretic peptide and B-type natriuretic peptide (BNP) by preventing their catabolism with neprilysin resulting in increase of cyclic guanosine monophosphate. This action of neprilysin is associated with increased natriuresis, diuresis, and systemic vasodilation, since these peptides have been shown to have potent diuretic, natriuretic, and vasodilating effects. In addition, it reduces the levels of N terminal pro-BNP. Therefore, administration of LCZ-696 results in significant reduction of wall stress from pressure and volume overload of the left ventricle as demonstrated by the reduction of N terminal pro-BNP, both significant constituents of hypertension and HF, and it is safe, well tolerated and is almost free of cough and angioedema. Copyright © 2017 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.

  7. Pharmacodynamic and Pharmacokinetic Profiles of Sacubitril/Valsartan (LCZ696) in Patients with Heart Failure and Reduced Ejection Fraction.

    Science.gov (United States)

    Kobalava, Zhanna; Kotovskaya, Yulia; Averkov, Oleg; Pavlikova, Elena; Moiseev, Valentine; Albrecht, Diego; Chandra, Priya; Ayalasomayajula, Surya; Prescott, Margaret F; Pal, Parasar; Langenickel, Thomas H; Jordaan, Pierre; Rajman, Iris

    2016-08-01

    Concomitant renin-angiotensin-aldosterone system blockade and natriuretic peptide system enhancement may provide unique therapeutic benefits to patients with heart failure and reduced ejection fraction (HFrEF). This study assessed the pharmacodynamics and pharmacokinetics of LCZ696 in patients with HFrEF. This was an open-label, noncontrolled single-sequence study. After a 24-h run-in period, patients (n = 30) with HFrEF (EF ≤ 40%; NYHA class II-IV) received LCZ696 100 mg twice daily (bid) for 7 days and 200 mg bid for 14 days, along with standard treatment for heart failure (HF) (except angiotensin-converting enzyme inhibitors [ACEIs] or angiotensin receptor blockers [ARBs]). On Day 21, significant increases were observed in the plasma biomarkers indicative of neprilysin and RAAS inhibition (ratio-to-baseline: cyclic guanosine monophosphate [cGMP], 1.38; renin concentration and activity, 3.50 and 2.27, respectively; all, P sacubitril (neprilysin inhibitor prodrug), LBQ657 (active neprilysin inhibitor), and valsartan were reached within 0.5, 2.5, and 2 h. Between 100- and 200-mg doses, the Cmax and AUC0-12 h for sacubitril and LBQ657 were approximately dose-proportional while that of valsartan was less than dose-proportional. Treatment with LCZ696 for 21 days was well tolerated and resulted in plasma biomarker changes indicative of neprilysin and RAAS inhibition in patients with HF. The pharmacokinetic exposure of the LCZ696 analytes in patients with HF observed in this study is comparable to that observed in the pivotal Phase III study. © 2016 John Wiley & Sons Ltd.

  8. Aphrodisiacs past and present: a historical review.

    Science.gov (United States)

    Sandroni, P

    2001-10-01

    The drug Viagra (sildenafil) has drawn public attention to aphrodisiacs. The search for such substances dates back millennia. Aphrodisiacs can be classified by their mode of action into 3 types: those that increase (1) libido, (2) potency, or (3) sexual pleasure. Various substances of animal and plant origin have been used in folk medicines of different cultures; some have been identified pharmacologically, allowing for understanding of their mechanisms of action. For increasing libido, ambrein, a major constituent of Ambra grisea, is used in Arab countries. This tricyclic triterpene alcohol increases the concentration of several anterior pituitary hormones and serum testosterone. Bufo toad skin and glands contain bufotenine (and other bufadienolides), a putative hallucinogenic congener of serotonin. It is the active ingredient in West Indian "love stone" and the Chinese medication chan su. The aphrodisiac properties are likely of central origin, as are the other effects of the drug. For increasing potency, Panax ginseng used in traditional Chinese medicine, works as an antioxidant by enhancing nitric oxide synthesis in the endothelium of many organs, including the corpora cavernosa; ginsenosides also enhance acetylcholine-induced and transmural nerve stimulation-activated relaxation associated with increased tissue cyclic guanosine monophosphate, hence the aphrodisiac properties. For increasing sexual pleasure, cantharidin ("Spanish fly") is a chemical with vesicant properties derived from blister beetles, which have been used for millennia as a sexual stimulant. Its mode of action is by inhibition of phosphodiesterase and protein phosphatase activity and stimulation of beta-receptors, inducing vascular congestion and inflammation. Morbidity from its abuse is significant. The ingestion of live beetles (Palembus dermestoides) in Southeast Asia and triatomids in Mexico may have a basis similar to cantharidin. It is of paramount importance for the physician to be

  9. MicroR828 regulates lignin and H2O2 accumulation in sweet potato on wounding.

    Science.gov (United States)

    Lin, Jeng-Shane; Lin, Chih-Ching; Lin, Hsin-Hung; Chen, Yu-Chi; Jeng, Shih-Tong

    2012-10-01

    MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. miRNAs play multiple roles in the growth, development and stress responses in plants. However, little is known of the wounding-responsive miRNAs and their regulation. Here, we investigated the expression patterns of microR828 (miR828) on wounding in sweet potato (Ipomoea batatas cv Tainung 57). The expression of miR828 was only detected in leaves, and was induced by wounding rather than by ethylene, hydrogen peroxide (H2O2), methyl jasmonate or nitric oxide (NO). Moreover, cyclic guanosine monophosphate (cGMP) was necessary for miR828 accumulation in leaves on wounding. Two miR828 target candidates, named IbMYB and IbTLD, were obtained by cDNA cloning, and their mRNA cleavage caused by miR828 was confirmed by cleavage site mapping, agro-infiltration and transgenics studies. The reduction in IbMYB and IbTLD expression coincided with the induction of miR828, demonstrating that IbMYB and IbTLD might be miR828 targets. Furthermore, transgenic sweet potato overexpressing miR828 precursor affected lignin and H2O2 contents. These results showed that cGMP could regulate wounding-responsive miR828, which repressed the expression of IbMYB and IbTLD. Subsequently, lignin and H2O2 were accumulated to participate in defense mechanisms. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  10. Effects of PDE5 Inhibitors and sGC Stimulators in a Rat Model of Artificial Ureteral Calculosis.

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    Peter Sandner

    Full Text Available Urinary colics from calculosis are frequent and intense forms of pain whose current pharmacological treatment remains unsatisfactory. New and more effective drugs are needed to control symptoms and improve stone expulsion. Recent evidence suggested that the Nitric Oxide (NO / cyclic guanosine monophosphate (cGMP/phosphodiesterase type 5 (PDE5 system may contribute to ureteral motility influencing stone expulsion. We investigated if PDE5 inhibitors and sGC stimulators influence ureteral contractility, pain behaviour and stone expulsion in a rat model of ureteral calculosis. We investigated: a the sex-specific PDE5 distribution in the rat ureter; b the functional in vitro effects of vardenafil and sildenafil (PDE5 inhibitors and BAY41-2272 (sGC stimulator on induced ureteral contractility in rats and c the in vivo effectiveness of vardenafil and BAY41-2272, alone and combined with ketoprofen, vs hyoscine-N-butylbromide alone or combined with ketoprofen, on behavioural pain indicators and stone expulsion in rats with artificial calculosis in one ureter. PDE5 was abundantly expressed in male and female rats' ureter. In vitro, both vardenafil and BAY41-2272 significantly relaxed pre-contracted ureteral strips. In vivo, all compounds significantly reduced number and global duration of "ureteral crises" and post-stone lumbar muscle hyperalgesia in calculosis rats. The highest level of reduction of the pain behaviour was observed with BAY41-2272 among all spasmolytics administered alone, and with the combination of ketoprofen with BAY41-2272. The percentage of stone expulsion was maximal in the ketoprofen+BAY41-2272 group. The NO/cGMP/PDE5 pathway is involved in the regulation of ureteral contractility and pain behaviour in urinary calculosis. PDE5 inhibitors and sGC stimulators could become a potent new option for treatment of urinary colic pain.

  11. Novel water-soluble curcumin derivative mediating erectile signaling.

    Science.gov (United States)

    Abdel Aziz, Mohamed Talaat; El Asmer, Mohammed F; Rezq, Ameen; Kumosani, Taha Abdullah; Mostafa, Samya; Mostafa, Taymour; Atta, Hazem; Abdel Aziz Wassef, Mohamed; Fouad, Hanan H; Rashed, Laila; Sabry, Dina; Hassouna, Amira A; Senbel, Amira; Abdel Aziz, Ahmed

    2010-08-01

    Curcumin is an inducer of heme oxygenase enzyme-1 (HO-1) that is involved in erectile signaling via elevating cyclic guanosine monophosphate (cGMP)levels. To assess the effect of oral administration of a water-soluble long-acting curcumin derivative on erectile signaling. Two hundred and thirty six male white albino rats were divided into four groups; group 1 (N = 20) includes control. Group 2 (N = 72) was equally divided into four subgroups; subgroup 1 received pure curcumin (10 mg/kg), subgroup 2 received the long-acting curcumin derivative (2 mg/kg), subgroup 3 received the long-acting curcumin derivative (10 mg/kg), and subgroup 4 received sildenafil (4 mg/kg). Subgroups were sacrificed after the first, second, and third hour. Group 3 (N = 72) was equally divided into the same four subgroups already mentioned and were sacrificed after 24 hours, 48 hours, and 1 week. Group 4 (N = 72) was subjected to intracavernosal pressure (ICP) measurements 1 hour following oral administration of the same previous doses in the same rat subgroups. Cavernous tissue HO enzyme activity, cGMP, and ICP. In group 2, there was a significant progressive maintained elevation of HO activity and cGMP tissue levels starting from the first hour in subgroups 3 and 4, whereas, the rise in HO activity and cGMP started from second hour regarding the other rat subgroups. Sildenafil effect decreased after 3 hours. In group 3, there was a significant maintained elevation of HO activity and cGMP tissue levels extended to 1 week as compared to controls for all rat subgroups that received both forms of curcumin. In group 4, long-acting curcumin derivative exhibited more significant potentiation of intracavernosal pressure as compared to control and to the pure curcumin. Water-soluble long-acting curcumin derivative could mediate erectile function via upregulating cavernous tissue cGMP. © 2009 International Society for Sexual Medicine.

  12. 3,7-Bis(2-hydroxyethyl)icaritin, a potent inhibitor of phosphodiesterase-5, prevents monocrotaline-induced pulmonary arterial hypertension via NO/cGMP activation in rats.

    Science.gov (United States)

    Lan, Tao-Hua; Chen, Xiao-Ling; Wu, Yun-Shan; Qiu, Hui-Liang; Li, Jun-Zhe; Ruan, Xin-Min; Xu, Dan-Ping; Lin, Dong-Qun

    2018-06-15

    Pulmonary arterial hypertension (PAH) is a chronic progressive disease which leads to elevated pulmonary arterial pressure and right heart failure. 3,7-Bis(2-hydroxyethyl)icaritin (ICT), an icariin derivatives, was reported to have potent inhibitory activity on phosphodiesterase type 5 (PDE5) which plays a crucial role in the pathogenesis of PAH. The present study was designed to investigate the effects of ICT on monocrotaline (MCT)-induced PAH rat model and reveal the underlying mechanism. MCT-induced PAH rat models were established with intragastric administration of ICT (10, 20, 40 mg/kg/d), Icariin (ICA) (40 mg/kg/d) and Sildenafil (25 mg/kg/d). The mean pulmonary arterial pressure (mPAP) and right ventricle hypertrophy index (RVHI) were measured. Pulmonary artery remodeling was assessed by H&E staining. Blood and lung tissue were collected to evaluate the level of endothelin 1 (ET-1), nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The expressions endothelial nitric oxide synthase (eNOS) and PDE5A in lung tissues were determined by Western blot analysis. The results showed that ICT reduced RVHI and mPAP, and reversed lung vascular remodeling in rats with MCT-induced PAH. ICT also reversed MCT-induced ET-1 elevation, NO and cGMP reduction in serum or lung tissue. Moreover, ICT administration significantly induced eNOS activation and PDE5A inhibition. ICT with lower dose had better effects than ICA. In summary, ICT is more effective in preventing MCT-induced PAH in rats via NO/cGMP activation compared with ICA. These findings demonstrate a novel mechanism of the action of ICT that may have value in prevention of PAH. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Organic Nitrate Therapy, Nitrate Tolerance, and Nitrate-Induced Endothelial Dysfunction: Emphasis on Redox Biology and Oxidative Stress.

    Science.gov (United States)

    Daiber, Andreas; Münzel, Thomas

    2015-10-10

    Organic nitrates, such as nitroglycerin (GTN), isosorbide-5-mononitrate and isosorbide dinitrate, and pentaerithrityl tetranitrate (PETN), when given acutely, have potent vasodilator effects improving symptoms in patients with acute and chronic congestive heart failure, stable coronary artery disease, acute coronary syndromes, or arterial hypertension. The mechanisms underlying vasodilation include the release of •NO or a related compound in response to intracellular bioactivation (for GTN, the mitochondrial aldehyde dehydrogenase [ALDH-2]) and activation of the enzyme, soluble guanylyl cyclase. Increasing cyclic guanosine-3',-5'-monophosphate (cGMP) levels lead to an activation of the cGMP-dependent kinase I, thereby causing the relaxation of the vascular smooth muscle by decreasing intracellular calcium concentrations. The hemodynamic and anti-ischemic effects of organic nitrates are rapidly lost upon long-term (low-dose) administration due to the rapid development of tolerance and endothelial dysfunction, which is in most cases linked to increased intracellular oxidative stress. Enzymatic sources of reactive oxygen species under nitrate therapy include mitochondria, NADPH oxidases, and an uncoupled •NO synthase. Acute high-dose challenges with organic nitrates cause a similar loss of potency (tachyphylaxis), but with distinct pathomechanism. The differences among organic nitrates are highlighted regarding their potency to induce oxidative stress and subsequent tolerance and endothelial dysfunction. We also address pleiotropic effects of organic nitrates, for example, their capacity to stimulate antioxidant pathways like those demonstrated for PETN, all of which may prevent adverse effects in response to long-term therapy. Based on these considerations, we will discuss and present some preclinical data on how the nitrate of the future should be designed.

  14. Effect of sildenafil citrate on secondary healing in full thickness skin defects in experiment.

    Science.gov (United States)

    Cakmak, E; Karasoy Yesilada, A; Sevim, K Z; Sumer, O; Tatlidede, H S; Sakiz, D

    2014-01-01

    An acceleration of the wound healing process expedites chronic wound patient's return to normal social environments significantly. Sildenafil, a cyclic guanosine monophosphate (cGMP)-dependent phosphodiesterase- 5 inhibitor has been shown to be a potent stimulator of angiogenesis through upregulation of cGMP. In our study, sildenafil was administered orally as a cost-effective supplement in the treatment of full thickness defects and chronic wounds in that manner with low incidence of side effects and morbidity. Randomly selected 72 Wistar-Albino rats were divided into the two groups, 36 rats in each group. Control group (n =36) was divided further into a secondary healing group consisting of 9 rats and a pathology group consisting of 27 rats (pathology group 1: 9 rats, 4th and 7th day of wound healing, pathology group 2: 9 rats, 10th and 14th day of wound healing, pathology group 3: 9 rats, 21st and 28th day of wound healing. Experimental group consisted of 36 rats which received sildenafil citrate (Viagra® Pfizer, Germany) for secondary wound healing to proceed. The average wound healing period in the control group was 17.89 days and in the sildenafil citrate administered group 14.56 days. The difference of the epithelialisation on full thickness defects were more prominent on days 5 and 11 postoperatively. In the sildenafil citrate applied group, on the 7th day, the defect was 25% smaller and on the 13th day, the defect contracted by 38%. In conclusion, we believe that sildenafil citrate administered orally is a cost- effective supplement in the treatment of full thickness defects and chronic wounds in that manner with low incidence of side effects and morbidity (Tab. 4, Fig. 7, Ref. 34).

  15. Clinical and preclinical treatment of urologic diseases with phosphodiesterase isoenzymes 5 inhibitors: an update

    Directory of Open Access Journals (Sweden)

    Wen-Hao Zhang

    2016-01-01

    Full Text Available Phosphodiesterase isoenzymes 5 inhibitors (PDE5-Is are the first-line therapy for erectile dysfunction (ED. The constant discoveries of nitric oxide (NO/cyclic guanosine monophosphate (cGMP cell-signaling pathway for smooth muscle (SM control in other urogenital tracts (UGTs make PDE5-Is promising pharmacologic agents against other benign urological diseases. This article reviews the literature and contains some previously unpublished data about characterizations and activities of PDE5 and its inhibitors in treating urological disorders. Scientific discoveries have improved our understanding of cell-signaling pathway in NO/cGMP-mediated SM relaxation in UGTs. Moreover, the clinical applications of PDE5-Is have been widely recognized. On-demand PDE5-Is are efficacious for most cases of ED, while daily-dosing and combination with testosterone are recommended for refractory cases. Soluble guanylate cyclase (sGC stimulators also have promising role in the management of severe ED conditions. PDE5-Is are also the first rehabilitation strategy for postoperation or postradiotherapy ED for prostate cancer patients. PDE5-Is, especially combined with α-adrenoceptor antagonists, are very effective for benign prostatic hyperplasia (BPH except on maximum urinary flow rate (Q max with tadalafil recently proved for BPH with/without ED. Furthermore, PDE5-Is are currently under various phases of clinical or preclinical researches with promising potential for other urinary and genital illnesses, such as priapism, premature ejaculation, urinary tract calculi, overactive bladder, Peyronie′s disease, and female sexual dysfunction. Inhibition of PDE5 is expected to be an effective strategy in treating benign urological diseases. However, further clinical studies and basic researches investigating mechanisms of PDE5-Is in disorders of UGTs are required.

  16. Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

    KAUST Repository

    Marondedze, Claudius

    2013-01-05

    Background: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. Findings. Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. Conclusions: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. 2013 Marondedze et al.; licensee BioMed Central Ltd.

  17. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade

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    Alexandre Dumoulin

    2018-04-01

    Full Text Available Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP, the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα. In the absence of any one of these components, neurons in dorsal root ganglia (DRG and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  18. Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

    Science.gov (United States)

    Rotcheewaphan, Suwatchareeporn; Belisle, John T; Webb, Kristofor J; Kim, Hee-Jin; Spencer, John S; Borlee, Bradley R

    2016-09-01

    The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

  19. Effect of quercetin on cadmium chloride-induced impairments in sexual behaviour and steroidogenesis in male Wistar rats.

    Science.gov (United States)

    Ujah, G A; Nna, V U; Agah, M I; Omue, L O; Leku, C B; Osim, E E

    2018-03-01

    Cadmium chloride (CdCl 2 ) has been reported to cause reproductive toxicity in male rats, mainly through oxidative stress. This study examined its effect on sexual behaviour, as one of the mechanisms of reproductive dysfunction, as well as the possible ameliorative effect of quercetin (QE) on same. Thirty male Wistar rats (10 weeks old), weighing 270-300 g, were used for this study. They were either orally administered 2% DMSO, CdCl 2 (5 mg/kg b.w.), QE (20 mg/kg b.w.) or CdCl 2 +QE, once daily for 4 weeks, before sexual behavioural studies. The 5th group received CdCl 2 for 4 weeks and allowed 4-week recovery period, before sexual behavioural test. Rats were sacrificed after sexual behavioural studies. The blood, testis and penis were collected for biochemical assays. Cadmium increased mount, intromission and ejaculatory latencies, but reduced their frequencies, compared to control. Serum nitric oxide increased, while penile cyclic guanosine monophosphate reduced in the CdCl 2 -exposed rats, compared to control. CdCl 2 increased testicular cholesterol, but reduced 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-HSD activities, and testosterone concentration. QE better attenuated these negative changes compared to withdrawal of CdCl 2 treatment. In conclusion, CdCl 2 suppressed steroidogenesis, penile erection and sexual behaviour, with poor reversal following withdrawal, while QE attenuated these effects. © 2017 Blackwell Verlag GmbH.

  20. Three molecular forms of atrial natriuretic peptides: quantitative analysis and biological characterization.

    Science.gov (United States)

    Nagai-Okatani, Chiaki; Kangawa, Kenji; Minamino, Naoto

    2017-07-01

    Atrial natriuretic peptide (ANP) is primarily produced in the heart tissue and plays a pivotal role in maintaining cardiovascular homeostasis in endocrine and autocrine/paracrine systems and has clinical applications as a biomarker and a therapeutic agent for cardiac diseases. ANP is synthesized by atrial cardiomyocytes as a preprohormone that is processed by a signal peptidase and stored in secretory granules as a prohormone. Subsequent proteolytic processing of ANP by corin during the secretion process results in a bioactive form consisting of 28 amino acid residues. Mechanical stretch of the atrial wall and multiple humoral factors directly stimulates the transcription and secretion of ANP. Secreted ANP elicits natriuretic and diuretic effects via cyclic guanosine monophosphate produced through binding to the guanylyl cyclase-A/natriuretic peptide receptor-A. Circulating ANP is subjected to rapid clearance by a natriuretic peptide receptor-C-mediated mechanism and proteolytic degradation by neutral endopeptidase. In humans, ANP is present as three endogenous molecular forms: bioactive α-ANP, a homodimer of α-ANP designated as β-ANP, and an ANP precursor designated as proANP (also referred to as γ-ANP). The proANP and especially β-ANP, as minor forms in circulation, are notably increased in patients with cardiac diseases, suggesting the utility of monitoring the pathophysiological conditions that result in abnormal proANP processing that cannot be monitored by inactive N-terminal proANP-related fragments. Emerging plate-based sandwich immunoassays for individual quantitation of the three ANP forms enables evaluation of diagnostic implications and net ANP bioactivity. This new tool may provide further understanding in the pathophysiology of cardiac diseases. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  1. Oxygen mediates vascular smooth muscle relaxation in hypoxia.

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    Jessica Dada

    Full Text Available The activation of soluble guanylate cyclase (sGC by nitric oxide (NO and other ligands has been extensively investigated for many years. In the present study we considered the effect of molecular oxygen (O2 on sGC both as a direct ligand and its affect on other ligands by measuring cyclic guanosine monophosphate (cGMP production, as an index of activity, as well as investigating smooth muscle relaxation under hypoxic conditions. Our isolated enzyme studies confirm the function of sGC is impaired under hypoxic conditions and produces cGMP in the presence of O2, importantly in the absence of NO. We also show that while O2 could partially affect the magnitude of sGC stimulation by NO when the latter was present in excess, activation by the NO independent, haem-dependent sGC stimulator 3-(5'-hydroxymethyl-2'-furyl-1-benzylindazole (YC-1 was unaffected. Our in vitro investigation of smooth muscle relaxation confirmed that O2 alone in the form of a buffer bolus (equilibrated at 95% O2/5% CO2 had the ability to dilate vessels under hypoxic conditions and that this was dependent upon sGC and independent of eNOS. Our studies confirm that O2 can be a direct and important mediator of vasodilation through an increase in cGMP production. In the wider context, these observations are key to understanding the relative roles of O2 versus NO-induced sGC activation.

  2. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

    Science.gov (United States)

    Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  3. The influence of Na+,K+-ATPase on glutamate signaling in neurodegenerative diseases and senescence

    Directory of Open Access Journals (Sweden)

    Paula Fernanda Kinoshita

    2016-06-01

    Full Text Available Decreased Na+,K+-ATPase (NKA activity causes energy deficiency, which is commonly observed in neurodegenerative diseases. The NKA is constituted of three subunits: α, β and γ, with four distinct isoforms of the catalytic α subunit (α1-4. Genetic mutations in the ATP1A2 gene and ATP1A3 gene, encoding the α2 and α3 subunit isoforms, respectively can cause distinct neurological disorders, concurrent to impaired NKA activity. Within the central nervous system (CNS, the α2 isoform is expressed mostly in glial cells and the α3 isoform is neuron-specific. Mutations in ATP1A2 gene can result in familial hemiplegic migraine (FHM2, while mutations in the ATP1A3 gene can cause Rapid-onset dystonia-Parkinsonism (RDP and alternating hemiplegia of childhood (AHC, as well as the cerebellar ataxia, areflexia, pescavus, optic atrophy and sensorineural hearing loss (CAPOS syndrome. Data indicates that the central glutamatergic system is affected by mutations in the α2 isoform, however further investigations are required to establish a connection to mutations in the α3 isoform, especially given the diagnostic confusion and overlap with glutamate transporter disease. The age-related decline in brain α2/3 activity may arise from changes in the cyclic guanosine monophosphate (cGMP and cGMP‐dependent protein kinase (PKG pathway. Glutamate, through nitric oxide synthase (NOS, cGMP and PKG, stimulates brain α2/3 activity, with the glutamatergic N-methyl-D-aspartate (NMDA receptor cascade able to drive an adaptive, neuroprotective response to inflammatory and challenging stimuli, including amyloid‐β. Here we review the NKA, both as an ion pump as well as a receptor that interacts with NMDA, including the role of NKA subunits mutations. Failure of the NKA-associated adaptive response mechanisms may render neurons more susceptible to degeneration over the course of aging.

  4. Gastro Hepatic Protective Effects of Sildenafil in γ-Irradiated Rats

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    Tawfik, S.S.; Salama, S.F.

    2013-01-01

    Sildenafil is a potent specific inhibitor of phosphodiesterase-5 (PDE-5), which ultimately increases intracellular cyclic guanosine monophosphate (cGMP) . Sildenafil commercially named Viagra; was studied for its gastro hepatic protective activity through acute exposure of rats to γ-rays. The experimental groups of rats were: Sildenafil [1 mg/ kg, intra venous (i.v.), in 0.2 ml saline] / day for 5 days and then exposed to 6 Gy γ-rays after 1 h of the last injection (sildenafil+ γ-rays group). Controls received saline as a vehicle/ for 5 day; sildenafil group received drug alone for 5 days, and γ-rays group received saline (without drug) for 5 days and exposed to 6 Gy γ-rays after 1 h of the last injection. All groups were decapitated on the 6th day. Gamma rays increased the level of malondialdehyde (MDA) and the activity of myeloperoxidase (MPO) but, lowered the levels of nitric oxide (NO) and reduced glutathione (GSH) as well as lowering the activities of superoxide dismutase (SOD) and catalase (CAT) in both stomach and hepatic tissues. Sildenafil administrated before γ-rays significantly reduced the level of MDA and the activity of MPO while elevating levels of NO and GSH plus activities of SOD and CAT in both stomach and hepatic tissues compared to control and sildenafil groups. Conclusion: The data reveals that sildenafil pre-treatment has a protective effect against γ-rays-induced gastro hepatic dysfunction and supports the possible use of sildenafil as a protective agent in γ-irradiated rats

  5. Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

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    Olinto, S.C.F. [Faculdade de Ciências Integradas do Pontal, Universidade Federal de Uberlândia, Ituiutaba, MG (Brazil); Adrião, M.G. [Departamento de Morfologia e Fisiologia, Universidade Federal Rural de Pernambuco, Recife, PE (Brazil); Castro-Barbosa, T.; Goulart-Silva, F.; Nunes, M.T. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

  6. Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

    Science.gov (United States)

    Dumoulin, Alexandre; Ter-Avetisyan, Gohar; Schmidt, Hannes; Rathjen, Fritz G

    2018-04-24

    Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

  7. Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rat.

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    Chao Liu

    Full Text Available Atrial natriuretic peptide (ANP provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A, is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of

  8. Nitric oxide in the nervous system: biochemical, developmental, and neurobiological aspects.

    Science.gov (United States)

    Cossenza, Marcelo; Socodato, Renato; Portugal, Camila C; Domith, Ivan C L; Gladulich, Luis F H; Encarnação, Thaísa G; Calaza, Karin C; Mendonça, Henrique R; Campello-Costa, Paula; Paes-de-Carvalho, Roberto

    2014-01-01

    Nitric oxide (NO) is a very reactive molecule, and its short half-life would make it virtually invisible until its discovery. NO activates soluble guanylyl cyclase (sGC), increasing 3',5'-cyclic guanosine monophosphate levels to activate PKGs. Although NO triggers several phosphorylation cascades due to its ability to react with Fe II in heme-containing proteins such as sGC, it also promotes a selective posttranslational modification in cysteine residues by S-nitrosylation, impacting on protein function, stability, and allocation. In the central nervous system (CNS), NO synthesis usually requires a functional coupling of nitric oxide synthase I (NOS I) and proteins such as NMDA receptors or carboxyl-terminal PDZ ligand of NOS (CAPON), which is critical for specificity and triggering of selected pathways. NO also modulates CREB (cAMP-responsive element-binding protein), ERK, AKT, and Src, with important implications for nerve cell survival and differentiation. Differences in the regulation of neuronal death or survival by NO may be explained by several mechanisms involving localization of NOS isoforms, amount of NO being produced or protein sets being modulated. A number of studies show that NO regulates neurotransmitter release and different aspects of synaptic dynamics, such as differentiation of synaptic specializations, microtubule dynamics, architecture of synaptic protein organization, and modulation of synaptic efficacy. NO has also been associated with synaptogenesis or synapse elimination, and it is required for long-term synaptic modifications taking place in axons or dendrites. In spite of tremendous advances in the knowledge of NO biological effects, a full description of its role in the CNS is far from being completely elucidated. © 2014 Elsevier Inc. All rights reserved.

  9. Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

    Science.gov (United States)

    Leite, Natália Rodrigues Pereira; Siqueira de Medeiros, Mariana; Mury, Wanda Vianna; Matsuura, Cristiane; Perszel, Monique Bandeira Moss; Noronha Filho, Gerson; Brunini, Tatiana Mc; Mendes-Ribeiro, Antônio Claúdio

    2016-08-01

    Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity. © 2016 John Wiley & Sons Australia, Ltd.

  10. Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

    Science.gov (United States)

    Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

    2018-03-01

    Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

  11. Involvement of NO-cGMP pathway in anti-hyperalgesic effect of PDE5 inhibitor tadalafil in experimental hyperalgesia.