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2010-01-01
The goal of this study was to examine whether the A3 adenosine receptor (A3AR) agonist Cl-IB-MECA protects against myocardial ischemia/reperfusion injury when administered at the time of reperfusion in an in vivo mouse model of infarction induced by 30min of coronary occlusion and 24h of reperfusion. Treating B6 wild-type with Cl-IB-MECA during the reperfusion phase (100mg/kg i.v. bolus+0.3mg/kg/min subcutaneously via implantation of Alzet mini-osmotic pumps) reduced myocardial infarct size ~37% from 50.1+/-2.5% in vehicle-treated mice to 31.6+/-2.8% in Cl-IB-MECA-treated mice, and significantly reduced the number of leukocytes that infiltrated into the ischemic-reperfused myocardium. Cl-IB-MECA did not reduce infarct size or limit leukocyte accumulation in studies using B6 congenic A3AR g...
2010-01-01
Abstract Aim: The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N6-(3-iodobenzyl)-adenosine-5prime-N-methylcarboxamide (IB-MECA) was studied. Methods: To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. Results: We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resi...
2010-01-01
Adenosine is released from injured or hypoxic tissues where it exerts numerous anti-inflammatory effects including suppression of neutrophil functions. Although most previous work has implicated the A2AAR, we have recently shown that selective activation of the abundantly expressed A3AR inhibits neutrophil superoxide production and chemotaxis providing a potential mechanistic explanation for the efficacy of A3AR agonists in experimental animal models of inflammation. In this study, we hypothesized that the A3AR suppresses neutrophil functions by inhibiting the monomeric GTPase Rac, a central regulator of chemokine-directed neutrophil migration and superoxide production. We found that pre-treating neutrophils with the highly selective A3AR agonist CP-532,903 reduced fMLP-induced Rac activat...
2010-01-01
A number of N6-substituted adenosine-5prime-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a-4f and 5a-5f, showed modest receptor affinity for the A3AR with Ki values in the low to mid mM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N6-position (in compounds 4g and 5g) significantly improved the affinity with Ki values of 30 and 9nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalised linker was introduced. The N6-phenyl series, compounds 7a-7d, demonstrated low to mid nanomolar receptor affinities (Ki=2.3-45.0nM), with 7b displaying 109-fold selectivity for the...
Adenosine A3 receptors regulate heart rate, motor activity and body temperature
2010-01-01
Abstract Aim: To examine the phenotype of mice that lack the adenosine A3 receptor (A3R). Methods: We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition, the effect of the adenosine analogue R-N6-phenylisopropyl-adenosine (R-PIA) was examined. We also examined heat production and food intake. Results: We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A3R knock-out mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R-PIA in wild type mice was virtually eliminated in the A3R knock-out mice. The marked reduction in activity was associated with a decreased heat prod...
2010-01-01
Desirability theory (DT) is a well-known multi-criteria decision-making approach. In this work, DT is employed as a prediction model (PM) interpretation tool to extract useful information on the desired trade-offs between binding and relative efficacy of N6-substituted-4prime-thioadenosines A3 adenosine receptor (A3AR) agonists. At the same time, it was shown the usefulness of a parallel but independent approach providing a feedback on the reliability of the combination of properties predicted as a unique desirability value. The appliance of belief theory allowed the quantification of the reliability of the predicted desirability of a compound according to two inverse and independent but complementary prediction approaches. This information is proven to be useful as a ranking criterion in ...
Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors
2010-01-01
In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A3 receptors activation. We revealed that A3 receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A3 receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A3 receptors, by inducing an ...
Chromosomal mapping of the mouse A3 adenosine receptor gene, Adora3
1995-11-01
A3 adenosine receptor is a member of Gi protein-coupled receptors that mediate inhibition of adenylate cyclase activity upon binding to the ligand. We determined the chromosome localization of the mouse A3 gene for future genetic studies, utilizing an interspecific backcross panel formed from the cross (C57BL/6J x Mus spretus)F{sub 1} x M. spretus. Genomic DNAs from 94 individuals in the backcross were analyzed by Southern hybridization with murine A3 receptor cDNA probe. Unique map positions were determined by haplotype analysis with 1388 previously mapped loci in the mouse backcross. The mouse A3 receptor gene (Adora3) mapped to chromosome 3, in tight linkage with DNA marker D3Bir15. 12 refs., 1 fig.
2010-02-01
We previously synthesized a series of potent and selective A{sub 3} adenosine receptor (AR) agonists (North-methanocarba nucleoside 5{prime}-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed 'click' chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A{sub 3}AR activation was preserved in these multivalent conjugates, which bound with apparent Ki of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A{sub 3}AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A{sub 3} and P2Y{sub 14} receptors (via amide-linked uridine-5{prime}-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.
2010-01-01
AbstractAim:To investigate whether adenosine A3 receptors (A3AR) stimulation restore vascular reactivity after hemorrhagic shock through a ryanodine receptor (RyR)-mediated and large conductance calcium-activated potassium (BKCa) channel-dependent pathway.Methods:Rat hemorrhagic shock model (40 mmHg) and vascular smooth muscle cell (VSMC) hypoxic model were used. The expression of A3AR was determined by Western blot and RT-PCR. The effect of A3AR stimulation on RyR-mediated Ca2+ release in VSMCs was analyzed by the Fura-3/AM loading Ca2+ imaging. The modulation of vascular reactivity to norepinephrine (NE) by A3AR stimulation was monitored by an isolated organ tension instrument.Results:Decrease of A3AR expression is consistent with the loss of vasoreactivity to NE in hemorrhagic shock rat...
2010-01-01
Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A1 and A2A adenosine receptors (ARs), but not of A2B and A3ARs. Activation of the heterologously expressed human A3AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H2O2 (750mM) but not in untransfected cells. The A3 agonist IB-MECA (EC50 3.8mM) and the non-selective agonist NECA (EC50 3.9mM) protected A3 AR-transfected cells against H2O2 in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendri...
http://hdl.handle.net/10072/5645
Adenosine is an important cardioprotective agent that works via several adenosine receptor (ADOR) subtypes to regulate cardiovascular activity. It is well established that functional responses to adenosine decline with age. What is unclear, though, is whether these changes occur at the receptor, second messenger or translational level. In this study we determined the effect of age on cardiac adenosine receptor expression using the housekeeping gene 18S rRNA versus the adenosine A2B receptor gene as internal controls. Absolute quantification showed that no age-related changes occurred in the expression of 18S rRNA or adenosine A2B receptor internal control genes. Subsequently, relative analysis of the adenosine receptor subtypes using 18S rRNA found a significant age-related reduction in the expression of the adenosine A1 receptor (5.5-fold), with no changes in the expression of the adenosine A2A, A2B and A3 receptors. When using the expression of the adenosine A2B receptor as the internal control gene, a significant down regulation of both the adenosine A1 (5.4-fold) and A2A (2.2-fold) receptors with no change in the expression of adenosine A3 receptor was found. Therefore, the high level of expression of the 18S rRNA housekeeping gene was found to mask a significant change in expression of the adenosine A2A receptor with age. Ultimately, these findings show an age-related reduction in adenosine A1 and A2A receptor expression in rat heart. Publisher: http://dx.doi.org/10.1016/j.mad.2003.11.016; Elsevier; Ireland; http://www.elsevier.com/wps/find/journaldescription.cws_home/506026/description#description Relation: Mechansims of Ageing and Development; 211; 217; 125 Other identifier: 0047-6374 Language: en_AU Rights: Copyright 2004 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links.
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060913.103656
Pyrazolo[3,4-d]pyrimidines were known as adenosine antagonists at the rat A1 and A2A adenosine receptors based on our previous studies. In this study, 245 pyrazolo[3,4-d]pyrimidines derivatives with various benzyl substitutents at N-1 and various hydrophobic side chains at C-4 and C-6 were synthesized and screened at the human A1, A2A and A3 adenosine receptors. 14 out of 245 compounds were resynthesized and purified to determine the Ki values of these compounds at the human A1 adenosine receptor. Chapter 1 of the thesis is a literature review of adenosine research. It describes the physiology of adenosine and the discovery and characterization of all adenosine receptors namely A1, A2A, A2B and A3. It also looks at the medical application of adenosine, adenosine analogs, adenosine agonists and adenosine antagonists. The final part of the chapter discusses the discovery and development of adenosine agonists and antagonists. Chapter 2 of the thesis describes the rational design of the pyrazolo[3,4-d]pyrimidines template using ligand-based molecular modelling technique and describes the synthesis of the template. Chapter 3 and chapter 4 describe the application of silicon chemistry and attempts to synthesise a series of pyrazolo[3,4-d]pyrimidines heterocycle by solid phase synthesis. Chapter 5 and chapter 6 describe the synthesis of a series of pyrazolo[3,4-d]pyrimidines heterocycle using the solution phase parallel synthesis and the binding studies of a library of 245 compounds and the resynthesis of 14 target compounds. Chapter 7 describes the cell culture and membrane preparation of the human A1, A2A, A2B and A3 adenosine receptors and radioligands binding assays. Publisher: Griffith University. School of Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Khoa Quang Do
2003-01-01
Elevated extracellular adenosine has been found to stimulate hematopoiesis in experimental mice exposed to radiotherapy (gamma-rays), chemotherapy (5-fluorouracil), or combined action of both these modalities (gamma-rays + carboplatin). These findings have been obtained after treatment of the animals with the combination of dipyridamole (DP), preventing the cellular uptake of adenosine, and adenosine monophosphate (AMP), acting as adenosine prodrug. Increased cycling of hematopoietic progenitor cells following the administration of DP + AMP has been shown to represent an important mechanism of acceleration of regeneration of suppressed hematopoiesis. In recent experiments, non-degradable synthetic adenosine receptor agonists, more or less specific for individual subtypes of adenosine receptors (A1, A2A, A2B, and A3 subtypes) have been studied. These ... >>
http://hdl.handle.net/10072/25829
Adenosine kinase phosphorylates adenosine to AMP, the primary pathway for adenosine metabolism under basal conditions. Inhibition of adenosine kinase results in a site–specific increase in interstitial adenosine. Using a rat model of myocardial infarction, we examined the protective effects of adenosine kinase inhibition. Male Sprague–Dawley rats underwent 30 min regional occlusion followed by 90 min reperfusion. Infarct size, expressed as a percent of the area–at–risk, IS/AAR(%), was 58.0 ± 2.1 % in untreated rats. Pretreatment with the adenosine kinase inhibitor, 5–iodotubercidin (1 mg/kg), limited infarct development to 37.5±3.7% (P < 0.001). The A1 adenosine receptor (A1AR) antagonist, DPCPX (100 µg/kg), abolished the infarct–sparing effect of 5–iodotubercidin (IS, 62.8 ± 1.3%). Similarly, the A3 adenosine receptor (A3AR) antagonist, MRS–1523 (2 mg/kg), and the δ–opioid receptor (DOR) antagonist, BNTX, (1 mg/kg) abolished the reduction of IS produced by iodotubercidin. Pretreatment with the ROS scavenger, 2–MPG (20 mg/kg), or the PKC–δ antagonist, rottlerin (0.3 mg/kg) also abolished iodotubercidin–mediated cardioprotection. Furthermore, pretreatment with 5–HD, a mitochondrial KATP (mitoKATP) channel inhibitor, but not the sarcolemmal KATP channel blocker, HMR–1098, abrogated the beneficial effects of adenosine kinase inhibition (IS, 59.5 ± 3.8%). These data suggest that inhibition of adenosine kinase is effective in reducing infarct development via A1AR, A3AR and DOR activation. Data also suggest that this protection is mediated via ROS, PKC–δ and mitoKATP channels. Publisher: http://dx.doi.org/10.1007/s00395-005-0526-7; Dr. Dietrich Steinkopff Verlag; Germany Relation: Basic Research in Cardiology; 328; 336; N; 100 Other identifier: 0300-8428 Language: en_AU Rights: Y
http://eprints.qut.edu.au/15127/
Herein we report the synthesis and biological evaluation of some potent and selective A1 adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N6-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (Ki = 50 nM) and good selectivity (A3/A1 400) for the A1 adenosine receptor. N6-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (Ki = 7 nM), but lower selectivity (A3/A1 = 3). All compounds bind weakly (Ki > 1 μM) to A2A and A2B receptors. The combination of A1 agonist activity and antioxidant activity has the potential to produce cardioprotective effects. Publisher: Elsevier Relation: DOI:10.1016/j.bmcl.2007.07.035; Gregg, Alison D. and Bottle, Steven E. and Devine, Shane M. and Figler, Heidi and Linden, Joel and White, Paul and Pouton, Colin W. and Urmaliya, Vijay and Scammells, Peter J. (2007) Dual acting antioxidant A1 adenosine receptor agonists. Bioorganic & Medicinal Chemistry Letters, 17(9). pp. 5437-5441. Format: application/pdf Rights: Copyright 2007 Elsevier; Reproduced in accordance with the copyright policy of the publisher.
1995-01-20
Adenosine receptors have been implicated as important mediators of a diversity of physiological processes throughout the body. These receptors are members of the G protein-coupled receptor superfamily, a class of cell-surface receptors that, when activated, couple to a heterotrimeric G protein complex to effect signal transduction. Four different subtypes of adenosine receptor have been identified through molecular cloning and subsequent pharmacological and biochemical analyses. Of these subtypes, the Al and A2a receptors have been mapped to chromosome 22q11.2-q13.1 and 11q11-q13, respectively. The A3 receptor has been localized to chromosome 3 in the mouse, by interspecific backcross analysis, suggesting a human chromosomal localization of 1p13 from known mouse human linkage homologies. In determining the chromosomal localization of the A2a receptor, a minor hybridization peak was detected on chromosome 10q25.3-q26.3, and the authors of this study concluded that this site was likely to correspond to an adenosine A2-like receptor. With the molecular identification of the adenosine A2b receptor, the hybridization site on chromosome 10 was assigned to the A2b receptor (MIM 102777). We have used fluorescence in situ hybridization (FISH) and PCR screening of a somatic cell hybrid panel to determine the true chromosomal localization of the A2b adenosine receptor subtype. 12 refs., 1 fig., 1 tab.
Localization of the adenosine A1 receptor subtype gene (ADORA1) to chromosome 1q32.1
1995-03-20
Adenosine, acting through its receptors, exerts effects on almost all organ systems, influencing a diversity of physiological responses, including the inhibition of neurotransmitter release, the modulation of cardiac rhythmicity and contractility, and the potentiation of IgE-dependent mediator release. Adenosine receptors belong to the G protein-coupled receptor superfamily, a class of cell-surface receptors that, when activated, couple to a heterotrimeric G protein complex to effect signal transduction. Molecular cloning and subsequent pharmacological and biochemical analyses have led to the identification of four different subtypes of adenosine receptor. The A3 receptor has been localized to chromosome 3 in the mouse by interspecific backcross analysis, suggesting a human chromosomal localization of 1p13 from known mouse-human linkage homologies. We have previously mapped the A2b adenosine receptor subtype to chromosome 17p11.2-p12 using fluorescence in situ hybridization (FISH) and PCR-based screening of somatic cell hybrid DNAs. A previous report has concluded that the Al and A2a receptor subtypes are localized on chromosome 22q11.2-q13.1 and 11q11-q13, respectively, but conflicts with that of MacCollin et al., who have mapped the A2a gene to chromosome 22. In this report, we show that the human A1 adenosine receptor subtype does not map to chromosome 22q11.2-q13.1, but is instead localized on chromosome 1q32. 13 refs., 1 fig.
http://eprints.jcu.edu.au/10022/1/aging_and_adenosine_transcription.pdf
The well-documented age-related change in ischemic tolerance may result from impaired adenosine-mediated cardioprotection. Additionally, ischemia itself may potentially modify adenosine signalling, contributing to the post-ischemic phenotype. This study investigates age- and ischemia-dependent changes in adenosine receptor transcript levels (Adora) for the A1, A2A, A2B, and A3 receptor subtypes in mouse myocardium. Hearts from young (2–4 months) and moderately aged (16–18 months) mice were subjected to 20-min ischemia and 45-min reperfusion. Ischemic tolerance was impaired in aged hearts, which recovered less than 30% ventricular pressure development (compared with ~70% in young hearts), and lost 2-fold higher levels of lactate dehydrogenase during reperfusion (reflecting cellular disruption). Real-time PCR analyses revealed an age-related decline in Adora3 levels and induction of Adora2B. Curiously, this effect was mimicked by ischemia, which acutely reduced Adora3 levels and induced Adora2B in young (but not old) hearts. In contrast, in aged hearts ischemia selectively reduced levels of Adora1 transcript (~2-fold) without altering transcript levels for the other receptors. These results demonstrate selective modulation of cardioprotective adenosine receptor transcription by both aging and ischemia. Reduced A3 adenosine receptor transcription may contribute to impaired ischemic tolerance in aged hearts, whereas changes in Adora transcription induced by ischemia may impact on the post-ischemic phenotype at later time points. Publisher: Elsevier Format: application/pdf Other identifier: Ashton, Kevin J., Nilsson, Ulrika, Willems, Laura, Holmgren, Kirsty, and Headrick, John P. (2003) Effects of aging and ischemia on adenosine receptor transcription in mouse myocardium. Biochemical and Biophysical Research Communications, 312 (2). pp. 367-372. ISSN 1090-2104
http://hdl.handle.net/10072/29336
Adenosine, a catabolite of ATP, exerts numerous effects in the heart, including modulation of the cardiac response to stress, such as occurs during myocardial ischemia and reperfusion. Over the past 20 years substantial evidence has accumulated that adenosine, administered either prior to ischemia or during reperfusion, reduces both reversible and irreversible myocardial injury. The latter effect results in reduction of both necrosis or myocardial infarction (MI) and apoptosis. These effects appear to be mediated via the activation of one or more G-protein coupled receptors (GPCRs), referred to as A1, A2A, A2B and A3 adenosine receptor (AR) subtypes. Experimental studies in different species and models suggest that activation of the A1 or A3ARs prior to ischemia is cardioprotective. Further experimental studies reveal that the administration of A2AAR agonists during reperfusion can also reduce MI, and recent reports suggest that A2BARs may also play an important role in modulating myocardial reperfusion injury. Despite convincing experimental evidence for AR-mediated cardioprotection, there have been only a limited number of clinical trials examining the beneficial effects of adenosine or adenosine-based therapeutics in humans, and the results of these studies have been equivocal. This review summarizes our current knowledge of AR-mediated cardioprotection, and the roles of the four known ARs in experimental models of ischemia-reperfusion. The chapter concludes with an examination of the clinical trials to date assessing the safety and efficacy of adenosine as a cardioprotective agent during coronary thrombolysis in humans. Publisher: Springer-Verlag; Berlin Contributor: CN Wilson, J Mustafa Relation: Handbook of Experimental Pharmacology, Adenosine Receptors in Health and Disease; 7; 19; 189; 214; N Other identifier: 978-3-540-89614-2 Language: en_AU Rights: Y
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060309.084525
Adenosine is a multi-functional physiological molecule found abundantly in the body. It is one of the important components of ATP cellular energy metabolism. Adenosine has diverse actions as a ligand on many different types of cells and tissues acting via specific receptors. Currently, four subtypes of adenosine receptors are described, namely, the A1, A2A, A2B and A3 receptors. Neuroblastoma, mostly found in young children, is a malignant tumor derived from peripheral neurons in the body. Several different types of neuroblastoma cell lines of human origin have been established and contributed to the studies of neuroblastoma itself, neuronal differentiation, neurotransmitters, alcoholism, Alzheimer's disease and other neuronal diseases and disorders. In 1987, it was shown by Abbracchio et al. that a human neuroblastoma cell line, IMR32, could be induced to differentiate into cells that have a more neuronal morphology, with long neurites, by an adenosine receptor agonist 5'-N-ethylcarboxamideadenosine (NECA) 2. 'Neuronal differentiation' is expected to be a new alternative to the conventional clinical therapies, such as surgery, chemotherapy and radiotherapy. Unlike IMR32, PC12 cells, a rat adrenal pheochromocytoma cell line, resembling human neuroblastoma cell lines and also expressing the A2 subtype of adenosine receptors, was shown not to differentiate under stimulation of the A2A subtype of adenosine receptors 3. Moreover, adenosine inhibited neuronal differentiation in mouse dorsal root ganglion cells presumably via the A1 subtype 4. The mechanism(s) of these confusing effects of adenosine on neuronal differentiation require examination. First, a detection method for each of the adenosine receptor subtypes was developed using reverse transcriptase polymerase chain reaction (RT-PCR). This provided a sensitive, non-radioactive, analytical tool. Subtype-specific, four pairs of PCR primers, corresponding to the A1, A2A, A2B and A3 receptors, were designed and synthesized. The RT-PCR study revealed the presence of adenosine A1, A2A and A2B receptor mRNAs in untreated SH-SY5Y cells. These PCR primers were also designed so that they would allow multiplex PCR. Optimization of conditions for multiplex PCR was conducted, allowing it to detect several adenosine receptor subtypes simultaneously, and it was proven to be partially successful. In the study of differentiation, the use of the designed PCR primers was not quantitative to measure the levels of adenosine receptors due to variations of the expressions levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, a house-keeping gene commonly used as the internal control in PCR or northern blot analysis. An adequate neuronal differentiation model system was established in order to study the possible role(s) of adenosine in neuronal differentiation. Nerve growth factor (NGF), a well-known inducer of differentiation of rat PC12 cells, did not show any apparent differentiation effects on human neuroblastoma SH-SY5Y cells. All-trans retinoic acid (50 µM) induced distinct neuronal differentiation in SH-SY5Y cells, however ethanol, used as a vehicle for retinoic acid, was also shown to have effects on this cell line causing morphological changes. Adenosine (100 µM) alone also did not induce marked differentiation in this cell line probably due to the presence of adenosine in serum. Adenosine deaminase-resistant, synthetic adenosine analogues were used and demonstrated enhancement of differentiation. A serum deprivation-induced differentiation in SH-SY5Y was found to be a consistent and useful model to evaluate the effects of other factors on differentiation in this cell line. This serum deprivation-induced differentiation was also found to accompany a substantial rise in the expression of neurofilament-H (NF-H), one of the marker proteins for neuronal differentiation, at the protein level. Using this model, the possible involvement of adenosine signaling via its receptors was investigated. Treatment of cells with selective adenosine analogues for the A1 and A2A subtypes, 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) and 2-[4-(2-carboxylethyl)phenylamino]-5'-N-ethylcarboxamido (CGS21680, 30 and 100 nM), respectively, enhanced the differentiation induced by serum deprivation at day 7 by approximately 60% and 70%, respectively. These enhancing effects of agonists were blocked by selective antagonists, 8-cyclophenyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinalzolin-5-amine (CGS15943), respectively. Simultaneous co-stimulation of the A1 and A2A subtypes with these agonists gave no further effects compared to the enhancing effects exerted by CCPA or CGS21680 alone. Signal transduction pathways were examined using various protein kinase inhibitors. A selective protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 100 nM) alone greatly enhanced the differentiation induced by serum deprivation in this cell line. No additive or synergistic effects of 10 nM H-89 with either the A1 or A2A receptor agonist were seen. A selective mitogen-activated protein kinase kinase (MAPKK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098,059) showed a similar pattern to H-89: 100 nM PD098,059 alone caused enhanced differentiation in serum deprivation-induced SH-SY5Y cells. The combination of PD098,059 and adenosine agonists did not show any further enhancement of differentiation. On the contrary, a selective protein kinase C (PKC) inhibitor, chelerythrine, suppressed the differentiation (by 51%) by serum deprivation at 1 uM, and at 100 nM, chelerythrine suppressed the enhancement of differentiation caused by CCPA and CGS21680 with no effect on the basic level of differentiation, indicating the possible involvement of PKC both in the differentiation induced by serum deprivation and the adenosine receptor-induced potentiation. Surprisingly, contrary to the assumption that the stimulation of PKA induces or assists neuronal differentiation, H-89 (20 uM) alone exerted a prompt differentiation (44% at day 2) in SH-SY5Y cells in the presence of the normal serum concentration (10%). This data suggests that the previously assumed role of PKA in differentiation must be re-evaluated. This H-89-induced differentiation model was shown to have a different differentiation mechanism to the previous serum deprivation-induced differentiation. Establishment of these new differentiation study models will add further options to explore neuronal differentiation, especially, of human type. Publisher: Griffith University. School of Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Rocky Hiroki Nishimura
2010-01-01
A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A1, A2A, A2B and A3 subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A2B and A1 receptors, with the most active compound (14q) having a pKi of 7.57 nM for hA2B receptors and a selectivity over hA2A receptors of 8.1-fold and hA1 receptors of 3.7-fold.
2010-01-01
G protein-coupled receptors (GPCRs) are therapeutic targets for many diseases, but progress in developing active and selective therapeutics has been severely hampered by the difficulty in obtaining accurate structures. We have been developing methods for predicting the structures for GPCR ligand complexes, but validation has been hampered by a lack of experimental structures with which to compare our predictions. We report here the predicted structures of the human adenosine GPCR subtypes (A1, A2A, A2B, and A3) and the binding sites for adenosine agonist and eight antagonists to this predicted structure, making no use of structural data, and compare with recent experimental crystal structure for ZM241385 bound human A2A receptor. The predicted structure correctly identifies 9 of the 12 cry...
http://hdl.handle.net/10072/29784
Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 μM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10-7M) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA; 10-7M) reduced the proportion of nonviable cells to 30.87 ± 2.49% and 35.18 ± 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 ± 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 ± 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 ± 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection. Publisher: http://dx.doi.org/10.1097/FJC.0b013e3181a443e2; Lippincott Williams & Wilkins; United States; http://journals.lww.com/cardiovascularpharm/pages/default.aspx Contributor: Michael R Rosen Relation: 5; Journal of Cardiovascular Pharmacology; 424; 433; Y; 53 Other identifier: 0160-2446 Language: en_AU Rights: Y
2010-01-01
Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a Ki value of 111+/-16nM in radioligand binding using [^3H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A2AAR antagonist. MRS5346 did not show any effect on A1 and A3 ARs in binding or the A2BAR in a cyclic AMP assay at 10mM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal follo...
[^1^8F]FEUPPY and [^1^8F]FEUPPY:2 metabolic considerations
2010-01-01
Introduction: Recently, [^1^8F]FEUPPY and [^1^8F]FEUPPY:2 were introduced as the first positron emission tomography (PET) tracers for the adenosine A3 receptor. Thus, aim of the present study was the metabolic characterization of the two adenosine A3 receptor PET tracers. Methods: In vitro carboxylesterase (CES) experiments were conducted using incubation mixtures containing different concentrations of the two substrates, porcine CES and phosphate-buffered saline. Enzymatic reactions were stopped by adding acetonitrile/methanol (10:1) after various time points and analyzed by a high-performance liquid chromatography (HPLC) standard protocol. In vivo experiments were conducted in male wild-type rats; tracers were injected through a tail vein. Rats were sacrificed after various time points (...
http://espace.library.uq.edu.au/view/UQ:158524
Purinergic receptors mediate a wide variety of biological effects in response to extracellular nucleotides and nucleosides, such as ATP, ADP, UTP, UDP and adenosine. To date, nineteen purinergic receptor subtypes have been identified and classified based on their sequence identity, pharmacological properties and signal transduction mechanisms and include the ionotropic P2X receptors (P2X1 - 7) and the G-protein coupled P2Y (P2Y1, 2, 4, 6, 12 - 14) and adenosine receptors (A1, A2a, A2b and A3). Purinergic receptor activation leads to changes in cytoplasmic [Ca2+] and/or [cAMP] and the modulation of mitogen activated protein kinase (MAP kinase) signalling cascades, which can result in mitogenic and/or inhibitory effects on cellular proliferation, migration and differentiation. In the central nervous system (CNS), extracellular ATP is released by neurons, astrocytes and vascular endothelial cells and plays a role in neurotransmission and neuromodulation. Furthermore, extracellular ATP acts as a critical signalling molecule for neuron-glial communication and is involved in the propagation of intercellular Ca2+ waves between astrocytes. The observation that purinergic signalling is an important factor in intercellular signalling, and has been shown to regulate proliferation, differentiation and survival in many cell types, has raised the possibility that purinergic receptors may also play a role in neurogenesis and development. Results presented in Chapter 3 demonstrate that undifferentiated neural progenitor cells isolated from the adult rat hippocampus express mRNA for all P2X receptor subtypes. The analysis of intracellular [Ca2+] transients in response to purinergic receptor agonists and other neurotransmitters show that hippocampal NPCs respond predominantly to ATP, ADP and glutamate via P2X7/P2X5 receptors, P2Y1 receptors and metabotropic glutamate receptors, respectively. Other neurotransmitters, such as GABA, glycine and acetylcholine were without effect. Electrophysiological measurements support these data and show that undifferentiated hippocampal NPCs share similar passive electrical properties to astrocytes, including electrical inexcitability and a high resting membrane potential (Q - 90 mV), providing a substantial electrochemical gradient for Ca2+ influx from the extracellular space via open P2X receptor ion channels. A more detailed analysis of purinergic receptor expression and function was conducted in primary neurosphere-derived NPCs generated in vitro from the subventricular zone (SVZ) of adult mice and in neural stem cells purified using flow cytometry (FACS) from acutely dissociated SVZ tissue. Results presented in Chapter 4 demonstrate that primary neurosphere-derived NPCs express similar purinergic receptors to FACS-purified neural stem cells. Both populations express mRNA for P2X4 and P2X7 receptors, all P2Y receptors, with the exception of the P2Y4 and the A1, A2a and A2b adenosine receptors. However, the neural stem cell population also expressed P2X1 and A3 receptor mRNA. ATP and the P2X7 receptor agonist, BzATP, evoked transient increases in cytoplasmic [Ca2+] in FACSpurified neural stem cells acutely dissociated from the SVZ, suggesting the functional expression of purinergic receptors. Furthermore, in similarity to NPCs isolated from the adult rat hippocampus, SVZ-derived NPCs exhibited transient increases in cytoplasmic [Ca2+] in response to purinergic and glutamatergic agonists but were insensitive to other neurotransmitters. Pharmacological profiling demonstrated that SVZ-derived NPCs express functional P2Y1 and P2Y2 receptors and a proportion of cells (Q 30%) showed transient increases in cytoplasmic [Ca2+] in response to the P2X7 receptor agonist BzATP, suggesting the functional expression of P2X7 and/or P2X4 receptors. Adenosine failed to elicit a Ca2+ response and did not significantly modulate basal levels of cAMP, however, A1 and A2a adenosine receptor protein was detected in primary neurospheres by western blot analysis. Having demonstrated that NPCs and neural stem cells derived from the SVZ of adult mice express functional purinergic receptors, we then investigated the effects of purinergic receptor activation on primary neurosphere frequency and proliferation in vitro. Experiments presented in Chapter 5 demonstrate that ATPyS, ADPbS and adenosine reduce the size and frequency of primary neurospheres in the presence of mitogens; epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), suggesting that crosstalk may occur between purinergic receptor and growth factor receptor signalling cascades associated with neural progenitor cell mitogenesis and survival. The inhibitory effect of ADPbS was antagonized by the P2Y1 receptor antagonist MRS 2179 and fully reversed by the P2Y12 receptor antagonist MRS 2395, which also antagonized adenosine receptors. The inhibitory effect of adenosine was attenuated by the A2a antagonist SCH 58261, and the A3 antagonist MRS 1523. Taken together, P2Y1, A2a and/or A3 receptors mediate inhibitory effects on growth factor-stimulated proliferation in primary neurospheres derived from the adult mouse SVZ. In passaged neurospheres, however, P2Y1 receptor activation potentiated NPC proliferation, whereas adenosine was inhibitory suggesting a change in ADP-sensitive P2Y receptor signalling occurs in NPCs selected for, and enriched over successive passages in the presence of mitogens EGF and bFGF. Neural progenitor cells have been reported to reside in a niche environment in close proximity to neurons astrocytes and the brain microvasculature. The functional expression of P2Y1, P2Y2 and P2X7 receptors in NPCs may enable these cells to participate in and/or respond to localized intercellular Ca2+ signalling within the neurogenic niche. Moreover, the modulation of neural stem cell and NPC proliferation by P2Y and adenosine receptor signalling pathways may represent a potential modulatory mechanism within the stem cell microenvironment (Stafford et al., in press).
2010-01-01
Adenosine receptors (ARs) belong to the G-protein-coupled receptor (GPCR) superfamily and consist of four subtypes referred to as A1, A2A, A2B, and A3. It is important to develop potent and selective modulators of ARs for therapeutic applications. In order to develop reliable in silico models that can effectively classify antagonists of each AR, we carried out three machine learning methods: Laplacian-modified naive Bayesian, recursive partitioning, and support vector machine. The results for each classification model showed values high in accuracy, sensitivity, specificity, area under the receiver operating characteristic curve and Matthews correlation coefficient. By highlighting representative antagonists, the models demonstrated their power and usefulness, and these models could be uti...
http://hdl.handle.net/10072/4736
The A3 adenosine receptor (A3AR) is attributed with multiple beneficial actions in ischemic–reperfused myocardium, including modulation of oncotic and apoptotic cell death and enhancement of contractile function. Additionally, the A3AR may attenuate vascular dysfunction and improve long-term outcome from myocardial insult (modulating hypertrophy and angiogenesis). Available evidence indicates that this receptor sub-type is minimally activated by endogenous adenosine during ischemia (A3AR antagonists exerting no effects on ischemic outcome), and is thus amenable to activation with exogenous agonists. Protected phenotypes arise with both pre- and post-ischemic treatment with A3AR agonists, and transient A3AR agonism also triggers early and delayed preconditioned states. The molecular basis for the varied protective actions of the A3AR remains poorly defined, and may well vary between species (e.g. rodent vs. human) and protective responses (e.g. acute vs. delayed protection). Nonetheless, A3ARs may be more promising as therapeutic "anti-ischemic" targets compared with other adenosine receptor subtypes, since A3AR agonists elicit fewer and less significant side-effects. This review addresses current knowledge and controversy regarding the protective actions (and associated signaling) of A3ARs in ischemic–reperfused heart. Publisher: http://dx.doi.org/10.1016/j.vph.2005.02.009; Elsevier; United States Relation: 5-6; Vascular Pharmacology; 271; 279; N; 42 Other identifier: 1537-1891 Language: en_AU Rights: Copyright 2005 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links; Y
Role of neutrophil purinergic receptors in organ dysfunction
2009-01-01
Neutrophils [polymorphonuclear leukocytes (PMNs)] express several purinergic receptors, including the nucleotide receptors P2Y2 and P2X7 and the adenosine receptor subtypes A1, A2A and A3. Activation of these receptors modulates PMN function and ultimately, in concert with other cells, affects the host's innate inflammatory response. PMN activities that can be altered by purinergic receptor stimulation include adhesion, aggregation, migration, phagocytosis, microbicidal function, release of tissue-damaging products and apoptosis. Interventions that alter PMN purinergic receptor stimulation are being developed to reduce organ dysfunction in conditions such as sepsis, ischemia–reperfusion injury and the acute respiratory distress syndrome.
http://hdl.handle.net/10072/6839
Objectives: To characterize effects of A3 adenosine receptor (A3AR) activation and gene knock-out on responses to ischemia-reperfusion in mouse heart. Methods: Perfused hearts from wild-type and A3AR gene knock-out (A3AR KO) mice were subjected to 20 min ischemia and 30 min reperfusion. Functional responses were assessed and changes in energy metabolism and cytosolic pH monitored via 31P-NMR spectroscopy. Results: Selective A3AR agonism with 100 nM 2-chloro-N6-(3-iodobenzyl)-adenosine-52-N-methyluronamide (chloro-IB-MECA) enhanced post-ischemic contractile recovery without altering contracture development in wild-type hearts, an effect unrelated to non-selective activation of A1 or A2 adenosine receptors. Chloro-IB-MECA also improved recovery in hearts overexpressing A1ARs. Paradoxically, post-ischemic recovery was enhanced by A3AR KO. Developed pressure, +dP/dt, and dP/dt all recovered to higher levels in A3AR KO (70 80% of pre-ischemia) vs. wild-type hearts (45 50% of pre-ischemia) (P<0.05). Enhanced recovery was unrelated to recoveries of ATP, phosphocreatine (PCr), inorganic phosphate (Pi), energy state ([ATP]/[ADP]. [Pi], GATP) or cytosolic pH. Conclusions: Selective A3AR activation is cardioprotective in wild-type hearts and hearts overexpressing A1ARs, yet A3AR gene deletion generates an ischemia-tolerant phenotype without altering energy metabolism or pH. This may be due to compensatory changes or undefined genotypic differences in A3AR KO vs. wild-type hearts. Publisher: Elsevier Science; Netherlands; http://www.elsevier.com/wps/find/journaldescription.cws_home/525398/description#description Relation: Cardiovascular Research; 147; 155; 53 Other identifier: 0008-6363 Language: en_AU Rights: Copyright 2003 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links.
Synthesis and biological evaluation of novel imidazole-containing macrocycles
2010-01-01
A new family of compounds made of a 5-aryl-1H-imidazole motif included in a macrocycle has been designed and synthesized. The synthesis of the imidazole core makes use of our previously developed method for the regioselective preparation of 1,2,5-trisubstituted imidazoles while the construction of the macrocycle is based on a three steps sequence: SNAr, Suzuki coupling, and RCM reaction. Biological evaluation of synthesized imidazole-containing macrocycles revealed that they display actual binding activity toward A3 adenosine (h) receptor, dopamine D1 (h) receptor, chloride channel (GABA-gated), and choline transporter (h) CHT1.
2010-01-01
A new series of 9-deazaxanthine derivatives with various substituents at the heterocyclic system were synthesized and evaluated for their binding affinities for the four human recombinant adenosine receptors, A1–A3 subtypes. A number of the 9-deazaxanthines derivatives 3a–m showed moderate-to-high affinity for hA2B receptors, with compound 3f showing a 32-fold selectivity for A2B over A1 and a 2750-fold selectivity for A2B over A2A.
http://hdl.handle.net/10072/6190
In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A1, A2A, A2B and A3, and estrogen receptors (ER) α and β. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA2A (>4-fold down) and consistent (>2-fold) down-regulation of ADORA1, ADORA3, and ER-β, following ovariectomy. No change was found in ADORA2B or ER-α. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain. Publisher: http://dx.doi.org/10.1016/S1385-299X(02)00219-2; Elsevier BV; Netherlands; http://www.elsevier.com/wps/find/journaldescription.cws_home/622287/description#description Contributor: F G Wouterlood Relation: Brain Research Protocols; 9; 18; 11 Other identifier: 1385-299X Language: en_AU Rights: Copyright 2003 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links
http://hdl.handle.net/10072/27908
We used pharmacological agents and genetic methods to determine whether the potent A3 adenosine receptor (AR) agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/ reperfusion injury in mice via the A3AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IBMECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 μg/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A3AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A2AAR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A3AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A3AR gene “knock-out” (A3KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A3KO mice in vivo and did not protect isolated perfused hearts obtained from A3KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of ρ-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IBMECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A3AR. Publisher: http://dx.doi.org/10.1124/jpet.106.111351; The American Society for Pharmacology and Experimental Therapeutics; Baltimore; http://jpet.aspetjournals.org/ Relation: 3; Journal of Pharmacology and Experimental Therapeutics; 1200; 1210; N; 319 Other identifier: 0022-3565 Language: en_AU Rights: Y
http://hdl.handle.net/10072/14270
This study evaluated the ability of A1 and A3 adenosine receptor (AR) agonism, and A1, A2A, A2B and A3AR antagonism (revealing "intrinsic" responses), to modify post-ischemic coronary dysfunction in mouse heart. Vascular function was assessed before and after 20 min global ischemia and 30-45 min reperfusion in Langendorff perfused C57/Bl6 mouse hearts. Ischemic insult impaired coronary sensitivity to the endothelial-dependent dilators ADP (pEC50=6.8+/-0.1 vs. 7.6+/-0.1, non-ischemic) and acetylcholine (pEC50=6.1+/-0.1 vs. 7.3+/-0.1 in non-ischemic), and for the mixed endothelial-dependent/independent dilator 2-chloroadenosine (pEC50=7.5+/-0.1 vs. 8.4+/-0.1, non-ischemic). Endothelium-independent dilation in response to nitroprusside was unaltered (pEC50=7.0+/-0.1 vs. 7.1+/-0.1 in non-ischemic). Pre-treatment with a selective A1AR agonist (50 nM CHA) failed to modify coronary dysfunction, whereas A1AR antagonism (200 nM DPCPX) worsened the effects of I/R (2-chloroadenosine pEC50=6.9+/-0.1). Conversely, A3AR agonism (100 nM Cl-IB-MECA) did reduce effects of I/R (pEC50s=8.0+/-0.1 and 7.3+/-0.1 for 2-chloroadenosine and ADP, respectively), whereas antagonism (100 nM MRS1220) was without effect. While A2AAR agonism could not be assessed (due to pronounced vasodilatation), A2AAR antagonism (100 nM SCH58261) was found to exert no effect, and antagonism of A2BARs (50 nM MRS1754) was also ineffective. The protective actions of A3AR agonism were also manifest as improved reactive hyperemic responses. Interestingly, post-ischemic coronary dysfunction was also limited by: Na+-H+ exchange (NHE) inhibition with 10 or 50 microM BIIB-513 (2-chloroadenosine pEC50s=7.8+/-0.1, either dose), an effect not additive with A3AR agonism; Ca2+ antagonism with 0.3 microM verapamil (2-chloroadenosine pEC50=7.9+/-0.1); and Ca2+ desensitization with 5 mM BDM (2-chloroadenosine pEC50=7.8+/-0.1). In contrast, endothelin antagonism (200 nM PD142893) and anti-oxidant therapy (300 microM MPG+150 U/ml SOD+600 U/ml catalase) were ineffective. Our data collectively confirm that ischemia selectively impairs endothelial function and reactive hyperemia independently of blood cells. Vascular injury is intrinsically limited by endogenous (but not exogenous) activation of A1ARs, whereas exogenous A3AR activation further limits dysfunction (improving post-ischemic vasoregulation). Finally, findings suggest this form of post-ischemic coronary injury is unrelated to endothelin or oxidant stress, but may involve modulation of Ca2+ overload and/or related ionic perturbations. Publisher: Elsevier Inc; New York, NY Relation: 2006; 21; Life sciences; 2426; 2437; N; 78 Other identifier: 0024-3205 Language: en_AU Rights: N
http://hdl.handle.net/2440/43863
Copyright © 2007 by the American Society for Pharmacology and Experimental Therapeutics.Originally published online on April 25th, 2007.The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A3 adenosine receptors (A3AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A3ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A3AR agonist N6-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A3AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(±)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A3AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport Vmax with no significant change in Km. As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK5H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A3ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A3ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.Chong-Bin Zhu, Jennifer A. Steiner, Jaclyn L. Munn, Lynette C. Daws, William A. Hewlett, and Randy D. Blakely Publisher: American Society for Pharmacology and Experimental Therapeutics Contributor: Clinical and Experimental Pharmacology Other identifier: Journal of Pharmacology and Experimental Therapeutics, 2007; 322 (1):332-340; 0022-3565; 0020076236; 10.1124/jpet.107.121665 Language: en
{sup 13}C relaxation in an RNA hairpin
1994-12-01
This initial survey of {sup 13}C relaxation in the {triangle}TAR RNA element has generated a number of interesting results that should prove generally useful for future studies. The most readily comparable study in the literature monitored {sup 13}C relaxation of the methyl groups from unusual bases in tRNA{sup Phe}. The study, which used T{sub 1} and NOE data only, reported order parameters for the methyl group axis that ranged between 0.51 and 0.97-a range similar to that observed here. However, they reported a breakdown of the standard order parameter analysis at higher (118-MHz {sup 13}C) frequencies, which should serve to emphasize the need for a thorough exploration of suitable motional models.
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20050830.120105
Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity. Publisher: Griffith University. School of Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Declan McKeveney
http://hdl.handle.net/2440/7750
None Available Other identifier: Neuroscience Research. 29(1):73-79; 0019971259 Language: en_US
http://hdl.handle.net/10072/6795
None Available Publisher: American Physiological Society; USA Relation: American Journal of Physiology: Heart and Circulatory Physiology; 793; 796; 282 Other identifier: 0363-6135 Language: en_AU
http://hdl.handle.net/10072/6137
None Available Publisher: JOHN WILEY & SONS INC; USA Relation: DRUG DEVELOPMENT RESEARCH; 447; 453; 58 Other identifier: 0272-4391 Language: en_AU
http://hdl.handle.net/10072/14104
None Available Publisher: Elsevier; Ireland Relation: Mechanisms of Ageing and Development; 264; 272; N; 127 Other identifier: 0047-6374 Language: en_AU Rights: Y
fPOP: footprinting functional pockets of proteins by comparative spatial patterns
2010-01-01
fPOP (footprinting Pockets Of Proteins, http://pocket.uchicago.edu/fpop/)...Full Text Available
fPOP: footprinting functional pockets of proteins by comparative spatial patterns
2010-01-01
Full Text Available.fPOP (footprinting Pockets Of Proteins, http://pocket.uchicago.edu/fpop/) is a relational database of the protein functional surfaces identified by analyzing the shapes of binding sites in ∼42 700 structures, including both holo and apo forms. We previously used a purely geometric method to extract the spatial patterns of functional surfaces (split pockets) in ∼19 000 bound structures and constructed a database, SplitPocket (http://pocket.uchicago.edu/). These functional surfaces are now used as spatial templates to predict the binding surfaces of unbound structures. To conduct a shape comparison, we use the Smith–Waterman algorithm to footprint an unbound pocket fragment with those of the functional surfaces in SplitPocket. The pairwise alignment of the unbound and bound pocket fragments is used to evaluate the local structural similarity via geometric matching. The final results of our large-scale computation, including ∼90 000 identified or predicted functional surfaces, are stored in fPOP. This database provides an easily accessible resource for studying functional surfaces, assessing conformational changes between bound and unbound forms and analyzing functional divergence. Moreover, it may facilitate the exploration of the physicochemical textures of molecules and the inference of protein function. Finally, our approach provides a framework for classification of proteins into families on the basis of their functional surfaces.
1994-12-01
The current emphasis in biological NMR work is on determining structures of biological macromolecules in solution. This emphasis is appropriate because NMR is the only technique capable of providing high-resolution structures that are comparable to those of x-ray crystallography for molecules in solution. This structural knowledge is immensely valuable and is needed in many areas of investigation. However, as valuable as such structural knowledge is, it never provides all the answers; a structure often reveals more questions than answers.
1994-12-01
Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.
Uncertainty analysis for absorbed dose from a brain receptor imaging agent
1999-01-01
Absorbed dose estimates are known to contain uncertainties. A recent literature search indicates that prior to this study no rigorous investigation of uncertainty associated with absorbed dose has been undertaken. A method of uncertainty analysis for absorbed dose calculations has been developed and implemented for the brain receptor imaging agent {sup 123}I-IPT. The two major sources of uncertainty considered were the uncertainty associated with the determination of residence time and that associated with the determination of the S values. There are many sources of uncertainty in the determination of the S values, but only the inter-patient organ mass variation was considered in this work. The absorbed dose uncertainties were determined for lung, liver, heart and brain. Ninety-five percent confidence intervals of the organ absorbed dose distributions for each patient and for a seven-patient population group were determined by the ``Latin Hypercube Sampling`` method. For an individual patient, the upper bound of the 95% confidence interval of the absorbed dose was found to be about 2.5 times larger than the estimated mean absorbed dose. For the seven-patient population the upper bound of the 95% confidence interval of the absorbed dose distribution was around 45% more than the estimated population mean. For example, the 95% confidence interval of the population liver dose distribution was found to be between 1.49E+0.7 Gy/MBq and 4.65E+07 Gy/MBq with a mean of 2.52E+07 Gy/MBq. This study concluded that patients in a population receiving {sup 123}I-IPT could receive absorbed doses as much as twice as large as the standard estimated absorbed dose due to these uncertainties.
Tuning microbial hosts for membrane protein production
The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition...Full Text Available
Tuning microbial hosts for membrane protein production
Full Text Available.The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months.This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production.
The Gene Wiki: community intelligence applied to human gene annotation
2010-01-01
Full Text Available.Annotating the function of all human genes is a critical, yet formidable, challenge. Current gene annotation efforts focus on centralized curation resources, but it is increasingly clear that this approach does not scale with the rapid growth of the biomedical literature. The Gene Wiki utilizes an alternative and complementary model based on the principle of community intelligence. Directly integrated within the online encyclopedia, Wikipedia, the goal of this effort is to build a gene-specific review article for every gene in the human genome, where each article is collaboratively written, continuously updated and community reviewed. Previously, we described the creation of Gene Wiki ‘stubs’ for approximately 9000 human genes. Here, we describe ongoing systematic improvements to these articles to increase their utility. Moreover, we retrospectively examine the community usage and improvement of the Gene Wiki, providing evidence of a critical mass of users and editors. Gene Wiki articles are freely accessible within the Wikipedia web site, and additional links and information are available at http://en.wikipedia.org/wiki/Portal:Gene_Wiki.
The Gene Wiki: community intelligence applied to human gene annotation
2010-01-01
Annotating the function of all human genes is a critical, yet formidable, challenge. Current gene annotation efforts focus on centralized curation resources, but it is increasingly clear that this approach...Full Text Available
Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the...Full Text Available
Full Text Available.Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor family for having a hypoxia-inducible factor (HIF1-α) binding site at its promoter region, and was dependent on an increase in the intracellular cAMP levels, but was independent of cAMP-dependent protein kinase (PKA). Further study of adenosine receptor signaling during intracellular bacterial infection could lead to breakthroughs in our understanding of persistent infections with these ubiquitous pathogens.
TNF-α and neuropathic pain - a review
Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous...Full Text Available
TNF-α and neuropathic pain - a review
Full Text Available.Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous system. Neuropathic pain is a recognized type of pathological pain where nociceptive responses persist beyond the resolution of damage to the nerve or its surrounding tissue. Very often, neuropathic pain is disproportionately enhanced in intensity (hyperalgesia) or altered in modality (hyperpathia or allodynia) in relation to the stimuli. At time of this writing, there is as yet no common consensus about the etiology of neuropathic pain - possible mechanisms can be categorized into peripheral sensitization and central sensitization of the nervous system in response to the nociceptive stimuli. Animal models of neuropathic pain based on various types of nerve injuries (peripheral versus spinal nerve, ligation versus chronic constrictive injury) have persistently implicated a pivotal role for TNF-α at both peripheral and central levels of sensitization. Despite a lack of success in clinical trials of anti-TNF-α therapy in alleviating the sciatic type of neuropathic pain, the intricate link of TNF-α with other neuro-inflammatory signaling systems (e.g., chemokines and p38 MAPK) has indeed inspired a systems approach perspective for future drug development in treating neuropathic pain.
1994-12-01
Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{sub 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.
Synthesis and NMR of {sup 15}N-labeled DNA fragments
1994-12-01
DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.
1994-12-01
Complete understanding of a protein`s function and mechanism of action can only be achieved with a knowledge of its three-dimensional structure at atomic resolution. At present, there are two methods available for determining such structures. The first method, which has been established for many years, is x-ray diffraction of protein single crystals. The second method has blossomed only in the last 5 years and is based on the application of nuclear magnetic resonance (NMR) spectroscopy to proteins in solution. This review paper describes three- and four-dimensional NMR methods applied to protein structure determination and was adapted from Clore and Gronenborn. The review focuses on the underlying principals and practice of multidimensional NMR and the structural information obtained.
Structure-Based Discovery of A2A Adenosine Receptor Ligands
2010-05-13
The recent determination of X-ray structures of pharmacologically relevant...Full Text Available
Structure-Based Discovery of A2A Adenosine Receptor Ligands
2010-05-13
Full Text Available.The recent determination of X-ray structures of pharmacologically relevant GPCRs has made these targets accessible to structure-based ligand discovery. Here we explore whether novel chemotypes may be discovered for the A2A adenosine receptor, based on complementarity to its recently determined structure. The A2A adenosine receptor signals in the periphery and the CNS, with agonists explored as anti-inflammatory drugs and antagonists explored for neurodegenerative diseases. We used molecular docking to screen a 1.4 million compound database against the X-ray structure computationally and tested 20 high-ranking, previously unknown molecules experimentally. Of these 35% showed substantial activity with affinities between 200 nM and 9 μM. For the most potent of these new inhibitors, over 50-fold specificity was observed for the A2A versus the related A1 and A3 subtypes. These high hit rates and affinities at least partly reflect the bias of commercial libraries toward GPCR-like chemotypes, an issue that we attempt to investigate quantitatively. Despite this bias, many of the most potent new ligands were novel, dissimilar from known ligands, providing new lead structures for modulation of this medically important target.
Structural studies on leukaemia inhibitory factor
1994-12-01
Leukaemia Inhibitory Factor (LIF) is a pleiotropic cytokine that acts on a wide range of target cells, including mega-karyocytes, osteoblasts, hepatocytes, adipocytes, neurons, embryonic stem cells, and primordial germ cells. Many of its activities are shared with other cytokines, particularly interleukin-6, oncostatin-M, ciliary neurotrophic factor, and granulocyte colony-stimulating factor (G-CSF). Although secreted in vivo as a glycoprotein, nonglycosylated recombinant protein expressed in E. coli is fully active and has been used in our nuclear magnetic resonance (NMR) studies of the three-dimensional structure and structure-function relationships of LIF. With 180 amino acids and a molecular mass of about 20 kDa, OF is too large for direct structure determination by two-dimensional and three-dimensional {sup 1}HNMR. It is necessary to label the protein with the stable isotopes {sup 15}N and {sup 13}C and employ heteronuclear three-dimensional NMR in order to resolve and interpret the spectral information required for three-dimensional structure determination. This work has been undertaken with both human LIF and a mouse-human chimaera that binds to the human LIF receptor with the same affinity as the human protein and yet expresses in E. coli at much higher levels. Sequence-specific resonance assignments and secondary structure elements for these proteins will be presented and progress towards determination of their three-dimensional structures described.
Skeletal Muscle Phenotypically Converts and Selectively Inhibits Metastatic Cells in Mice
Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with...Full Text Available
Skeletal Muscle Phenotypically Converts and Selectively Inhibits Metastatic Cells in Mice
Full Text Available.Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.
Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury
2010-05-01
Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation...Full Text Available
Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury
2010-05-01
Full Text Available.Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (A2BAR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9±3.4-fold, p<0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS-exposure. Studies utilizing A2BAR promoter constructs and RNA-protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR-bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.
Selective {sup 2}H and {sup 13}C labeling in NMR analysis of solution protein structure and dynamics
1994-12-01
Preparation of samples bearing combined isotope enrichment patterns has played a central role in the recent advances in NMR analysis of proteins in solution. In particular, uniform {sup 13}C, {sup 15}N enrichment has made it possible to apply heteronuclear multidimensional correlation experiments for the mainchain assignments of proteins larger than 30 KDa. In contrast, selective labeling approaches can offer advantages in terms of the directedness of the information provided, such as chirality and residue type assignments, as well as through enhancements in resolution and sensitivity that result from editing the spectral complexity, the relaxation pathways and the scalar coupling networks. In addition, the combination of selective {sup 13}C and {sup 2}H enrichment can greatly facilitate the determination of heteronuclear relaxation behavior.
Full Text Available.BackgroundChronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood.It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.MethodsDiseased (n = 13, n = 15 and n = 12 in IBD, psoriasis and RA respectively) and healthy patients (n = 18) were recruited based on strict inclusion and exclusion criteria; peripheral blood samples were collected by clinicians (30 ml) in Venous Blood Vacuum Collection Tubes containing EDTA and peripheral blood mononuclear cells were separated by Ficoll gradient centrifugation. RNA was extracted using Trizol reagent. Gene expression data was obtained using TaqMan Low Density Array (TLDA) containing 96 genes that were selected by an algorithm and the statistical analyses were performed in Prism by using non-parametric Mann-Whitney U test (P-values < 0.05).ResultsHere we show that using a panel of 96 disease associated genes and measuring mRNA expression levels in peripheral blood derived mononuclear cells; we could identify disease-specific gene panels that separate each disease from healthy controls. In addition, a panel of five genes such as ADM, AQP9, CXCL2, IL10 and NAMPT discriminates between all samples from patients with chronic inflammation and healthy controls. We also found genes that stratify the diseases and separate different subtypes or different states of prognosis in each condition.ConclusionsThese findings and the identification of five universal markers of chronic inflammation suggest that these diseases have a common background in pathomechanism, but still can be separated by peripheral blood gene expression. Importantly, the identified genes can be associated with overlapping biological processes including changed inflammatory response. Gene panels based on such markers can play a major role in the development of personalized medicine, in monitoring disease progression and can lead to the identification of new potential drug targets in chronic inflammation.
BackgroundChronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions...Full Text Available
Nonlinear dynamics of cardiovascular ageing
2010-03-01
Full Text Available.AbstractThe application of methods drawn from nonlinear and stochastic dynamics to the analysis of cardiovascular time series is reviewed, with particular reference to the identification of changes associated with ageing. The natural variability of the heart rate (HRV) is considered in detail, including the respiratory sinus arrhythmia (RSA) corresponding to modulation of the instantaneous cardiac frequency by the rhythm of respiration. HRV has been intensively studied using traditional spectral analyses, e.g. by Fourier transform or autoregressive methods, and, because of its complexity, has been used as a paradigm for testing several proposed new methods of complexity analysis. These methods are reviewed. The application of time–frequency methods to HRV is considered, including in particular the wavelet transform which can resolve the time-dependent spectral content of HRV. Attention is focused on the cardio-respiratory interaction by introduction of the respiratory frequency variability signal (RFV), which can be acquired simultaneously with HRV by use of a respiratory effort transducer. Current methods for the analysis of interacting oscillators are reviewed and applied to cardio-respiratory data, including those for the quantification of synchronization and direction of coupling. These reveal the effect of ageing on the cardio-respiratory interaction through changes in the mutual modulation of the instantaneous cardiac and respiratory frequencies. Analyses of blood flow signals recorded with laser Doppler flowmetry are reviewed and related to the current understanding of how endothelial-dependent oscillations evolve with age: the inner lining of the vessels (the endothelium) is shown to be of crucial importance to the emerging picture. It is concluded that analyses of the complex and nonlinear dynamics of the cardiovascular system can illuminate the mechanisms of blood circulation, and that the heart, the lungs and the vascular system function as a single entity in dynamical terms. Clear evidence is found for dynamical ageing.
Nonlinear dynamics of cardiovascular ageing
2010-03-01
AbstractThe application of methods drawn from nonlinear and stochastic dynamics to the analysis of cardiovascular time series is reviewed, with particular reference to the identification...Full Text Available
New strategy for stable-isotope-aided, multidimensional NMR spectroscopy of DNA oligomers
1994-12-01
Nuclear Magnetic Resonance (NMR) is the most efficient method for determining the solution structures of biomolecules. By applying multidimensional heteronuclear NMR techniques to {sup 13}C/{sup 15}N-labeled proteins, we can determine the solution structures of proteins with molecular mass of 20 to 30kDa at an accuracy similar to that of x-ray crystallography. Improvements in NMR instrumentation and techniques as well as the development of protein engineering methods for labeling proteins have rapidly advanced multidimensional heteronuclear NMR of proteins. In contrast, multidimensional heteronuclear NMR studies of nucleic acids is less advanced because there were no efficient methods for preparing large amounts of labeled DNA/RNA oligomers. In this report, we focused on the chemical synthesis of DNA oligomers labeled at specific residue(s). RNA oligomers with specific labels, which are difficult to synthesize by the enzyme method, can be synthesized by the chemical method. The specific labels are useful for conformational analysis of larger molecules such as protein-nucleic acid complexes.
Magnetic resonance studies of isotopically labeled paramagnetic proteins: (2FE-2S) ferredoxins
1994-12-01
Recent developments in NMR spectroscopy, especially multidimensional, multinuclear NMR techniques, have made NMR the most versatile tool available for studying protein structure and function in solution. Unlike diamagnetic proteins, paramagnetic proteins contain centers with unpaired electrons. These unpaired electrons interact with magnetic nuclei either through chemical bonds by a contact mechanism or through space by a pseudocontact mechanism. Such interactions make the acquisition and analysis of NMR spectra of paramagnetic proteins more challenging than those of diamagnetic proteins. Some NMR signals from paramagnetic proteins are shifted outside the chemical shift region characteristic of diamagnetic proteins; these {open_quotes}hyperfine-shifted{close_quotes} resonances originate from nuclei that interact with unpaired electrons from the paramagnetic center. The large chemical shift dispersion in spectra of paramagnetic proteins makes it difficult to excite the entire spectral window and leads to distortions in the baseline. Interactions with paramagnetic centers shorten T{sub 1} and T{sub 2} relaxation times of nuclei; the consequences are line broadening and lower spectral sensitivity. Scalar (through bond) and dipolar (through space) interactions between pairs of nuclei are what give rise to crosspeak signals in multi-dimensional NMR spectra of small diamagnetic proteins. When such interactions involve a nucleus that is strongly relaxed by interaction with a paramagnetic center, specialized methods may be needed for its detection or it may be completely undetectable by present nD NMR methods.
2010-01-01
Stimulation of adenosine A2A receptors results in anti-inflammatory effects in a variety of cell types. Lipopolysaccharide (LPS) and pro-inflammatory cytokines, such as TNF-a and IL-1, have been reported to up-regulate the expression of adenosine A2A receptors and thereby enhance the functional activity of adenosine A2A receptors in human and murine monocyte/macrophage cell lines and in monocytes/macrophages isolated from those species. In this study, we investigated the effects of LPS and TNF-a on the expression and functional activity of adenosine A2A receptors in isolated equine peripheral blood monocytes. The results of this study indicate that LPS and TNF-a up-regulate the transcription of adenosine A2A receptors for up to 24h; the response to LPS was of greater magnitude than the res...
2010-06-01
Full Text Available.Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1+/+) mice efficiently cleave BC. Bcmo1−/− mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1−/− mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1−/− mice and Bcmo1+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1−/− mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1−/− mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1−/− mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.
2010-06-01
Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice accumulate beta-carotene (BC) similarly...Full Text Available
1994-12-01
This paper deals with compounds that are chiral-at least in part, due to isotope substitution-and their use in tracing the steric course of enzyme reaction in vitro and in vivo. There are other applications of isotopically chiral compounds (for example, in analyzing the steric course of nonenzymatic reactions and in probing the conformation of biomolecules) that are important but they will not be discussed in this context.
Isotope labeling for NMR studies of macromolecular structure and interactions
1994-12-01
Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.
Increased epithelial stem cell traits in advanced endometrial endometrioid carcinoma
Full Text Available.BackgroundIt has been recognized cancer cells acquire characters reminiscent of those of normal stem cells, and the degree of stem cell gene expression correlates with patient prognosis. Lgr5(+) or CD133(+) epithelial stem cells (EpiSCs) have recently been identified and these cells are susceptible to neoplastic transformation. It is unclear, however, whether genes enriched in EpiSCs also contribute in tumor malignancy. Endometrial endometrioid carcinoma (EEC) is a dominant type of the endometrial cancers and is still among the most common female cancers. Clinically endometrial carcinoma is classified into 4 FIGO stages by the degree of tumor invasion and metastasis, and the survival rate is low in patients with higher stages of tumors. Identifying genes shared between advanced tumors and stem cells will not only unmask the mechanisms of tumor malignancy but also provide novel therapeutic targets.ResultsTo identify EpiSC genes in late (stages III-IV) EECs, a molecular signature distinguishing early (stages I-II) and late EECs was first identified to delineate late EECs at the genomics level. ERBB2 and CCR1 were genes activated in late EECs, while APBA2 (MINT2) and CDK inhibitor p16 tumor suppressors in early EECs. MAPK pathway was significantly up in late EECs, indicating drugs targeting this canonical pathway might be useful for treating advanced EECs. A six-gene mini-signature was further identified to differentiate early from advanced EECs in both the training and testing datasets. Advanced, invasive EECs possessed a clear EpiSC gene expression pattern, explaining partly why these tumors are more malignant.ConclusionsOur work provides new insights into the pathogenesis of EECs and reveals a previously unknown link between adult stem cells and the histopathological traits of EECs. Shared EpiSC genes in late EECs may contribute to the stem cell-like phenotypes shown by advanced tumors and hold the potential of being candidate therapeutic targets and novel prognosis biomarkers.
Increased epithelial stem cell traits in advanced endometrial endometrioid carcinoma
BackgroundIt has been recognized cancer cells acquire characters reminiscent of those of normal stem cells, and the degree of stem cell gene expression correlates with patient prognosis....Full Text Available
1994-12-01
To understand the details of macromolecular function, high-resolution structural and dynamic detail is essential. The polypeptide fold of the gramicidin channel has been effectively modeled for the past 20 years, yet the functional changes in conductance and channel lifetime associated with amino acid substitutions cannot be predicted. To accomplish this goal, high-resolution electrostatic modeling and the precise orientation of all dipoles are required. Furthermore, an enhanced knowledge of the complex molecular environment of this membrane-bound peptide is needed. An aqueous environment is relatively uniform and achiral. The membrane environment is very heterogenous and chiral. A knowledge of the interactions, specific and nonspecific, between peptide and lipid will aid in developing a better understanding of this environment. To accomplish this goal, it is necessary to study the peptide in an extended lipid bilayer, rather than in a vesicular or micellar form. These latter environments are likely to possess increased dynamics, increased water penetration, and distorted interactions between the polypeptide and membrane surface. To perform NMR studies on bilayer bound peptides, solid state NMR methods are required, and for specific site information, isotopic labels are incorporated using solid phase peptide synthesis.
Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma
PurposePreviously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study,...Full Text Available
Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma
Full Text Available.PurposePreviously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study, we studied the genes regulated by Ep-CAM in a retinoblastoma Y79 cell line in vitro using a combination of short interference RNA and microarray technology.MethodsFlow cytometry, quantitative reverse transcriptase PCR (Q-RT–PCR), and immunohistochemistry were performed to confirm the Ep-CAM re-expression in the Y79 cells treated with 5′-azacytidine (AZC). Ep-CAM expression in AZC-treated Y79 cells was silenced using synthetic anti-Ep-CAM short interference RNA, and whole genome microarray was performed to determine the gene expression changes post Ep-CAM knockdown. Ep-CAM inhibition was confirmed by Q-RT–PCR, western blotting, and immunofluorescence.ResultsEp-CAM expression was significantly restored in Y79 cells on day 5 of AZC treatment. Ep-CAM inhibition significantly affected Y79 cell proliferation. We identified 465 upregulated genes (≥1.0 fold) and 205 downregulated genes (≤0.5 fold) in response to knockdown of Ep-CAM. These genes regulate several aspects of tumor function, including cell survival/proliferation, DNA replication/transcription, apoptosis, and angiogenesis. Quantitative pathway analysis using Biointerpreter further revealed that the most pronounced effect of Ep-CAM knockdown was deregulation of pathways that include mitogen-activated protein kinase (MAP) kinase and tumor protein 53 (P53) pathways. Real-time Q-RT–PCR confirmed microarray gene expression changes for selected genes.ConclusionsEp-CAM silencing significantly decreases Y79 cell proliferation and revealed a wide network of deregulated pathways in vitro. Future studies targeting Ep-CAM gene expression in vivo will help to delineate the mechanisms associated with Ep-CAM gene function in neoplastic transformation and define the potential for Ep-CAM-based molecular intervention in retinoblastoma patients.
2010-01-01
Proteins of the G-protein coupled receptor (GPCR) family present numerous attractive targets for rational drug design, but also a formidable challenge for identification and conformational modeling...Full Text Available
2010-01-01
Full Text Available.Proteins of the G-protein coupled receptor (GPCR) family present numerous attractive targets for rational drug design, but also a formidable challenge for identification and conformational modeling of their 3D structure. A recently performed assessment of blind predictions of adenosine A2a receptor (AA2AR) structure in complex with ZM241385 (ZMA) antagonist provided a first example of unbiased evaluation of the current modeling algorithms on a GPCR target with ~30% sequence identity to the closest structural template. Several of the 29 groups participating in this assessment exercise (Michino et al., doi:10.1038/nrd2877) successfully predicted the overall position of the ligand ZMA in the AA2AR ligand binding pocket, however models from only three groups captured more than 40% of the ligand-receptor contacts.Here we describe two of these top performing approaches, in which all-atom models of the AA2AR were generated by homology modeling followed by ligand guided backbone ensemble receptor optimization (LiBERO). The resulting AA2AR-ZMA models, along with the best models from other groups are assessed here for their virtual ligand screening (VLS) performance on a large set of GPCR ligands.We show that ligand guided optimization was critical for improvement of both ligand-receptor contacts and VLS performance as compared to the initial raw homology models. The best blindly predicted models performed on par with the crystal structure of AA2AR in selecting known antagonists from decoys, as well as from antagonists for other adenosine subtypes and AA2AR agonists. These results suggest that despite certain inaccuracies, the optimized homology models can be useful in the drug discovery process.
Full Text Available.BackgroundThromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA2-induced Ca2+ influx, which resulted in vascular contraction via Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells.Methodology/Principal FindingsApplication of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by an L-type Ca2+ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+ ([Ca2+]i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.Conclusions/SignificanceThese data suggest a functional role of CNG channels in U-46619-induced Ca2+ influx and contraction of smooth muscle cells.
BackgroundThromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can...Full Text Available
1994-12-01
The conformations of two diazocine turn mimics, which were later incorporated into GPIIb/IIIa peptide antagonists, were investigated using nuclear magnetic resonance techniques. The two compounds, methyl (2,5-dioxo-3-(S)-(3-{omega}-tosylguanidino-propyl)-4-methyl-octahydro-1,4-dazocin-1-yl)acetate (1) and methyl (2,5-dioxo-3-(S)-(3-{omega}-tosyl-guanidino-propyl)-octahydro-1,5-diazocin-1-yl)acetate (2), differ only in their substituent at the diazocine position 4 nitrogen, yet this substitution results in a marked difference in the affinity of the resulting analogs for the GPIIb/IIIa receptor. It was of interest to determine if the difference observed in the antagonistic potency between these analogs was related to constitutional or, perhaps, conformational differences. The backbone conformations of these two molecules can be determined by measuring vicinal coupling constants along the trimethylene portion of the C8 ring backbone and by measuring interproton NOE intensities between the diazocine methine proton and the protons of the trimethylene group. For compound 1, {sup 3}J{sub HH} values measured from a P.E.COSY spectrum and interproton distances calculated from ROESY buildup curves indicated the presence of a single C8 ring backbone conformation where the trimethylene bridge adopted a staggered conformation and the H{alpha}1 and H{gamma}1 protons of the trimethylene group were 2.2 A from the methine proton. For compound 2, however, partial overlap of the central H{beta}1 and H{beta}2 protons made it impossible to measure {sup 3}J{sub HH} values from the P.E.COSY spectrum. We therefore used a {sup 13}C-filtered TOCSY experiment to measure the {sup 3}J{sub CH} values in both compounds 1 and 2. These heteronuclear vicinal coupling constants measured with {sup 13}C in natural abundance in conjunction with measured interproton NOE intensities indicate that these compounds share a common C8 ring backbone conformation.
Complex DNA structures and structures of DNA complexes
1994-12-01
Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.
CD73 represses pro-inflammatory responses in human endothelial cells
Full Text Available.BackgroundCD73 is a 5'-ectonucleotidase that produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors to induce cellular responses. CD73 has been reported to regulate expression of pro-inflammatory molecules in mouse endothelium. Our aim is to determine the function of CD73 in human endothelial cells.MethodsWe used RNAi to deplete CD73 levels in human umbilical cord endothelial cells (HUVECs).ResultsCD73 depletion resulted in a strong reduction in adenosine production, indicating that CD73 is the major source of extracellular adenosine in HUVECs. We find that CD73 depletion induces a similar response to pro-inflammatory stimuli such as the cytokine TNF-α. In CD73-depleted cells, surface levels of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin increase. This correlates with increased translocation of the transcription factor NF-kB to the nucleus, which is known to regulate ICAM-1, VCAM-1 and E-selectin expression in response to TNF-α. Adhesion of monocytic cells to endothelial cells is enhanced. In addition, CD73-depleted cells become elongated, have higher levels of stress fibres and increased endothelial permeability, resembling known responses to TNF-α.ConclusionsThese results indicate that CD73 normally suppresses pro-inflammatory responses in human endothelial cells.
CD73 represses pro-inflammatory responses in human endothelial cells
BackgroundCD73 is a 5'-ectonucleotidase that produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors to induce cellular responses. CD73 has been...Full Text Available
Adaptation of NS cells growth and differentiation to high-throughput screening-compatible plates
Full Text Available.BackgroundThere is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.ResultsWe report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays.ConclusionsOur results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.
Adaptation of NS cells growth and differentiation to high-throughput screening-compatible plates
BackgroundThere is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring...Full Text Available
2008-01-01
Searching for drugs conforming to requirements for protection and/or treatment of radiation-induced damage belongs to the most important tasks of current radiobiology. In the Laboratory of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic, two original approaches for stimulation of radiation-suppressed hematopoiesis have been tested in recent years, namely activation of adenosine receptors and inhibition of cyclooxygenases. Non-selective activation of adenosine receptors, induced by combined administration of dipyridamole, a drug preventing adenosine uptake and supporting thus its extracellular receptor-mediated action, and adenosine monophosphate, an adenosine prodrug, has been found to stimulate hematopoiesis when the drugs were given either pre- or post-irradiation. When ... >>
2010-03-01
Full Text Available.
Adenosine receptor subtypes in airways responses of sensitized guinea-pigs to inhaled ovalbumin
2010-01-01
Endogenous adenosine is released in asthmatic patients’ lungs by inhaled allergen, however, its exact role in asthmatic responses or the receptors mediating these responses has not been determined. Our hypothesis was that adenosine released during allergen challenge contributes to the airways responses to inhaled allergen. The effects of selective antagonists of the four adenosine receptor subtypes were investigated on the airways responses of sensitized guinea-pigs to inhaled ovalbumin to ascertain the role of adenosine in these allergen responses, and compared with a corticosteroid, dexamethasone. Early (EAR) and late asthmatic responses (LAR) to inhaled ovalbumin (10 mg/ml) of sensitized, conscious guinea-pigs were recorded by whole body plethysmography following administration o...
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