Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A(2A) receptors and redistribution of synaptic vesicles

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A(2A) receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [H-3]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A(2A) receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (03 mM)-induced transmitter release facilitation, because its effect was prevented by the A(2A) receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M-1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M-2 and Al receptors blocked by methoctramine (0.1 mu M) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A(2A) receptors by endogenous adenosine leading to synaptic vesicle redistribution. (C) 2014 Elsevier Ltd. All rights reserved.

Synthesis and preliminary evaluation of N-oxide derivatives for the prevention of atherothrombotic events

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspergillus fumigatus calcineurin interacts with a nucleoside diphosphate kinase

The Ca2+-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoH(V83F), was constructed, which demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoH(V83F) mutation did not cause a loss in virulence in the Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Structural and kinetic studies of Schistosoma mansoni adenylate kinases

The human parasite Schistosoma mansoni is totally dependent on the purine salvage pathway in order to supply large quantities of purine precursors for its energy and DNA biosynthetic needs. Adenylate kinase (ADK) is responsible for the conversion of AMP (produced by the adenosine kinase reaction) into ADP, which is subsequently converted into ATP by nucleoside diphosphate kinase (NDPK). ADK and NDPK are the most active enzymes of the pathway, probably reflecting an evolutionary adaptation due to the intense use of the branch in which they participate. However, notwithstanding their importance very little information has been accumulated found regarding these enzymes. In this work two adenylate kinases from S. mansoni were cloned and heterologously expressed in Escherichia coil. The purified products were utilized in activity assays, and displayed kinetic parameters similar to the corresponding human orthologous proteins. The cytosolic S. mansoni ADK was crystallized and its structure solved allowing us to detect a difference in the nucleotide binding site when compared with the human ortholog. (C) 2012 Elsevier B.V. All rights reserved.

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxy) ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43*) side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl-6 and Br-8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90) by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91)) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: Role for the adenosine A(2A) receptor

Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20 mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4 days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2[7-Amino-2-(2-furyl)[1,2,4] triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor. (C) 2012 Elsevier B. V. All rights reserved.

Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Acute adenosine increases cardiac vagal and reduces sympathetic efferent nerve activities in rats

Adenosine is the first drug of choice in the treatment of supraventricular arrhythmias. While the effects of adenosine on sympathetic nerve activity (SNA) have been investigated, no information is available on the effects on cardiac vagal nerve activity (VNA). We assessed in rats the responses of cardiac VNA, SNA and cardiovascular variables to intravenous bolus administration of adenosine. In 34 urethane-anaesthetized rats, cardiac VNA or cervical preganglionic sympathetic fibres were recorded together with ECG, arterial pressure and ventilation, before and after administration of three doses of adenosine (100, 500 and 1000 mu g kg-1). The effects of adenosine were also assessed in isolated perfused hearts (n= 5). Adenosine induced marked bradycardia and hypotension, associated with a significant dose-dependent increase in VNA (+204 +/- 56%, P < 0.01; +275 +/- 120%, P < 0.01; and +372 +/- 78%, P < 0.01, for the three doses, respectively; n= 7). Muscarinic blockade by atropine (5 mg kg-1, i.v.) significantly blunted the adenosine-induced bradycardia (-56.0 +/- 4.5%, P < 0.05; -86.2 +/- 10.5%, P < 0.01; and -34.3 +/- 9.7%, P < 0.01, respectively). Likewise, adenosine-induced bradycardia was markedly less in isolated heart preparations. Previous barodenervation did not modify the effects of adenosine on VNA. On the SNA side, adenosine administration was associated with a dose-dependent biphasic response, including overactivation in the first few seconds followed by a later profound SNA reduction. Earliest sympathetic activation was abolished by barodenervation, while subsequent sympathetic withdrawal was affected neither by baro- nor by chemodenervation. This is the first demonstration that acute adenosine is able to activate cardiac VNA, possibly through a central action. This increase in vagal outflow could make an important contribution to the antiarrhythmic action of this substance.

Value of adenosine infusion for infarct size determination using real-time myocardial contrast echocardiography

Abstract Background Myocardial contrast echocardiography has been used for determination of infarct size (IS) in experimental models. However, with intermittent harmonic imaging, IS seems to be underestimated immediately after reperfusion due to areas with preserved, yet dysfunctional, microvasculature. The use of exogenous vasodilators showed to be useful to unmask these infarcted areas with depressed coronary flow reserve. This study was undertaken to assess the value of adenosine for IS determination in an open-chest canine model of coronary occlusion and reperfusion, using real-time myocardial contrast echocardiography (RTMCE). Methods Nine dogs underwent 180 minutes of coronary occlusion followed by reperfusion. PESDA (Perfluorocarbon-Exposed Sonicated Dextrose Albumin) was used as contrast agent. IS was determined by RTMCE before and during adenosine infusion at a rate of 140 mcg·Kg-1·min-1. Post-mortem necrotic area was determined by triphenyl-tetrazolium chloride (TTC) staining. Results IS determined by RTMCE was 1.98 ± 1.30 cm2 and increased to 2.58 ± 1.53 cm2 during adenosine infusion (p = 0.004), with good correlation between measurements (r = 0.91; p < 0.01). The necrotic area determined by TTC was 2.29 ± 1.36 cm2 and showed no significant difference with IS determined by RTMCE before or during hyperemia. A slight better correlation between RTMCE and TTC measurements was observed during adenosine (r = 0.99; p < 0.001) then before it (r = 0.92; p = 0.0013). Conclusion RTMCE can accurately determine IS in immediate period after acute myocardial infarction. Adenosine infusion results in a slight better detection of actual size of myocardial damage.

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Untersuchungen zur Beeinflussung der Reparatur oxidativer DNA-Schäden durch Poly(ADP-Ribose)-Polymerase, AP-Endonuklease 1 und das Xeroderma pigmentosum A Protein

Gegenstand dieser Arbeit war die Untersuchung der Bedeutung der Poly(ADP-Ribose)-Polymerase 1 (PARP 1), der AP Endonuklease 1 (Ape 1) und des Xeroderma pigmentosum A (XPA) Proteins für die DNA-Reparatur in Säugerzellen.Zunächst wurde der Einfluss der PARP 1-Aktivität auf die Reparatur verschiedener DNA-Modifikationen untersucht. Die Ergebnisse zeigen erstmalig, dass eine Hemmung der PARP-Aktivität nicht nur eine deutliche Verlangsamung der Reparatur von Einzelstrangbrüchen, sondern auch von oxidativen Purinmodifikationen und Pyrimidindimeren zur Folge hat. Interessanterweise erfolgte diese Verlangsamung der DNA-Reparatur nicht in Csb-defizienten Zellen. Diese Ergebnisse deuten darauf hin, dass die Aktivierung der PARP 1 und das Csb-Protein zusammen an einem neuartigen Mechanismus beteiligt sind, der die globale Reparatur verschiedener DNA-Modifikationen beschleunigt.Weiterhin wurde die Bedeutung der Nukleotidexcisionsreparatur als back-up Reparatur von 8 Hydroxyguanin untersucht. Dazu wurden normale und XPA-defiziente Fibroblasten des Menschen mit einem hOgg1-anitsense Konstrukt transfiziert und dann in diesen Zellen die Reparaturkinetiken oxidativer Basenmodifikationen bestimmt. Dadurch konnte eine Beteiligung des XPA-Proteins an diesem Reparaturweg ausgeschlossen werden.Außerdem wurden die Auswirkungen einer AP Endonuklease-1-Überexpression in XRCC1-defizienten Zellen auf die Reparatur von Einzelstrangbrüchen untersucht. Die Reparatur der induzierten Einzelstrangbrüche war in XRCC1-defizienten Zellen erwartungsgemäß deutlich langsamer als in XRCC1-profizienten Zellen. Die Überexpression der AP Endonuklease 1 in XRCC1-defizienten Zellen führte zu einer teilweisen Beschleunigung der Einzelstrangbruchreparatur.

Synthesis of novel ribose-based thioimidate n-oxides

This work is based on the study of new synthetic paths to obtain thioimidate N-oxides (TINOs) from D-ribose and to study their reactivity with the purpose to obtain ketonitrones. TINOs, aren’t well known molecules, but these enantiomerically pure backbones could be valuable intermediates in the synthesis of novel ketonitrones which are key intermediates in the synthesis of iminosugars. TINOs were discovered from the study of glucoraphanin, a particular glucosinolate, that unexpectedly cyclized into a TINO after desulfatation, by a spontaneous intramolecular Michael addition. The first part of this work was to synthetize the TINO 3 from D-ribose 1. The key step was the desilylative cyclisation of a suitably functionalized thiohydroximate 2. Based on precedent work developed in the laboratory, we could obtain the thiohydroximate from D-ribose. We then focused our studies on the cyclisation step trying to find the suitable substituents that could give the TINO in good yield by desilylative cyclisation. The second part of the project is to obtain ketonitrones 4 by palladiumcatalyzed coupling reaction.

Quantum Mechnical Investion of Cyclic 3',5'- Adenosine Monophosphate, the Second Hormonal Messenger

Full geometry optimizations using the PM3, AM1, 3-21G∗/HF and 6-31G∗/HF levels of theory were conducted on the syn and anti conformations of cyclic3′,5′-adenosine monophosphate (cAMP). Comparison of the anti crystal structures with the semiempirical and ab initio results revealed that the ab initio results agree well with the experimental results. The results of semiempirical calculations are in qualitative agreement with experimental and ab initio values, with the exception of the glycosyl torsion angle for the anti conformer. Sugar puckering, which is not handled properly by semiempirical methods for unconstrained sugars, nucleosides, nucleotides and nucleotide base pairs, is modeled reasonably well by the semiempirical methods for cAMP. This improvement results from the constraints introduced by the cyclization of AMP to form the phosphodiester.

Use of the Supermolecule Approach To Model the Syn and Anti Conformations of Solvated Cyclic 3‘,5‘-Adenosine Monophosphate

The supermolecule approach has been used to model the hydration of cyclic 3‘,5‘-adenosine monophosphate, cAMP. Model building combined with PM3 optimizations predict that the anti conformer of cAMP is capable of hydrogen bonding to an additional solvent water molecule compared to the syn conformer. The addition of one water to the syn superstructure with concurrent rotation of the base about the glycosyl bond to form the anti superstructure leads to an additional enthalpy of stabilization of approximately −6 kcal/mol at the PM3 level. This specific solute−solvent interaction is an example of a large solvent effect, as the method predicts that cAMP has a conformational preference for the anti isomer in solution. This conformational preference results from a change in the number of specific solute−solvent interactions in this system. This prediction could be tested by NMR techniques. The number of waters predicted to be in the first hydration sphere around cAMP is in agreement with the results of hydration studies of nucleotides in DNA. In addition, the detailed picture of solvation about this cyclic nucleotide is in agreement with infrared experimental results.