Home About us Contact
|
Home About us Contact | ||
|
|
||
Competition Binding (competition + binding)
Selected Abstracts,9 -Tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult ratsEPILEPSIA, Issue 8 2010Andrew J. Hill Summary Purpose:, We assessed the anticonvulsant potential of the phytocannabinoid ,9 -tetrahydrocannabivarin (,9 -THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. Methods:, ,9 -THCV was applied before (10 ,m,9 -THCV) or during (10,50 ,m,9 -THCV) epileptiform activity induced by Mg2+ -free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of ,9 -THCV on CB1 receptors were examined using [3H]SR141716A competition binding and [35S]GTP,S assays in rat cortical membranes. Effects of ,9 -THCV (0.025,2.5 mg/kg) on pentylenetetrazole (PTZ),induced seizures in adult rats were also assessed. Results:, After induction of stable spontaneous epileptiform activity, acute ,9 -THCV application (,20 ,m) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 ,m,9 -THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, ,9 -THCV acted as a CB1 receptor ligand, displacing 0.5 nm [3H]SR141716A with a Ki,290 nm, but exerted no agonist stimulation of [35S]GTP,S binding. In PTZ-induced seizures in vivo, 0.25 mg/kg ,9 -THCV significantly reduced seizure incidence. Discussion:, These data demonstrate that ,9 -THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor,mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states. [source] Pigment epithelium-derived factor binds to cell-surface F1 -ATP synthaseFEBS JOURNAL, Issue 9 2010Luigi Notari Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a , 60 kDa PEDF-binding protein in bovine retina with Bos taurus F1 -ATP synthase ,-subunit, and that F1Fo -ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F1. Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real-time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of , 60 kDa. Antibodies to F1,-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F1, these results show that PEDF is a ligand for endothelial cell-surface F1Fo -ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. Structured digital abstract ,,MINT-7711286: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase delta subunit (uniprotkb:Q12165) and F-ATPase epsilon subunit (uniprotkb:P21306) by competition binding (MI:0405) ,,MINT-7711113: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit(uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) ,,MINT-7711060: F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase alpha subunit (uniprotkb:P07251) and PEDF (uniprotkb:P36955) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711313: F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), PEDF (uniprotkb:P36955), F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase gamma subunit(uniprotkb:P38077) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711083: PEDF (uniprotkb:P36955) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) [source] Definition of the residues required for the interaction between glycine-extended gastrin and transferrin in vitroFEBS JOURNAL, Issue 17 2009Suzana Kovac Transferrin is the main iron transport protein found in the circulation, and the level of transferrin saturation in the blood is an important indicator of iron status. The peptides amidated gastrin(17) (Gamide) and glycine-extended gastrin(17) (Ggly) are well known for their roles in controlling acid secretion and as growth factors in the gastrointestinal tract. Several lines of evidence, including the facts that transferrin binds gastrin, that gastrins bind ferric ions, and that the level of expression of gastrins positively correlates with transferrin saturation, suggest the possible involvement of the transferrin,gastrin interaction in iron homeostasis. In the present work, the interaction between gastrins and transferrin has been characterized by surface plasmon resonance and covalent crosslinking. First, an interaction between iron-free apo-transferrin and Gamide or Ggly was observed. The fact that no interaction was observed in the presence of the chelator EDTA suggested that the gastrin,ferric ion complex was the interacting species. Moreover, removal of ferric ions with EDTA reduced the stability of the complex between apo-transferrin and gastrins, and no interaction was observed between Gamide or Ggly and diferric transferrin. Second, some or all of glutamates at positions 8,10 of the Ggly molecule, together with the C-terminal domain, were necessary for the interaction with apo-transferrin. Third, monoferric transferrin mutants incapable of binding iron in either the N-terminal or C-terminal lobe still bound Ggly. These findings are consistent with the hypothesis that gastrin peptides bind to nonligand residues within the open cleft in each lobe of transferrin and are involved in iron loading of transferrin in vivo. Structured digital abstract ,,MINT-7212832, MINT-7212849: Apo-transferrin (uniprotkb:P02787) and Gamide (uniprotkb:P01350) bind (MI:0407) by surface plasmon resonance (MI:0107) ,,MINT-7212881, MINT-7212909: Ggly (uniprotkb:P01350) and Apo-transferrin (uniprotkb:P02787) bind (MI:0407) by cross-linking studies (MI:0030) ,,MINT-7212864: Apo-transferrin (uniprotkb:P02787) and Ggly (uniprotkb:P01350) bind (MI:0407) by competition binding (MI:0405) [source] Tristetraprolin recruits functional mRNA decay complexes to ARE sequencesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007Heidi H. Hau Abstract AU-rich elements (AREs) in the 3, untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF-alpha 3, UTR promoted TTP-dependent mRNA decay when it was inserted into the 3, UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5,,3, exonuclease Xrn1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript. J. Cell. Biochem. 100: 1477,1492, 2007. © 2006 Wiley-Liss, Inc. [source] Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemiaJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 4 2006C. I. BRANDON The aim of the current study was to clone the equine adenosine A2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA2A -R expression construct was generated by ligation of the eA2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA2A -R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (KD), and receptor densities (Bmax) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p -sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A2A -R agonists revealed that the eA2A -R functionally coupled to G,s as indicated by an increase in intracellular [3H]cAMP upon receptor activation. Finally, NF- ,B reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF- ,B activity. These results indicate that the heterologously expressed eA2A -R has a pharmacological profile similar to that of other mammalian A2A receptors and thus can be utilized for further characterization of the eA2A -R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease. [source] Substance P and CGRP expression in dental pulps with irreversible pulpitisAUSTRALIAN ENDODONTIC JOURNAL, Issue 2 2010Mandana Sattari dds Abstract The purpose of this study was to compare substance P (SP) and calcitonin gene-related peptide (CGRP) expression in pulp tissue with clinically diagnosed symptomatic and asymptomatic irreversible pulpitis. Healthy pulps acted as controls. Five normal pulps and 40 with irreversible pulpitis (20 symptomatic and 20 asymptomatic) were obtained from 45 different patients. SP and CGRP expression was determined by competition binding assays using enzyme immunoassay. anova and Mann,Whitney tests were used to ascertain if there were statistically significant differences between the groups. The results showed that neuropeptides were found in all pulp samples. The highest and the lowest expressions for SP and CGRP were found in symptomatic irreversible pulpitis and healthy pulps groups, respectively. The differences between healthy pulps and the groups of pulps having irreversible pulpitis were significant (P < 0.001). Although Mann,Whitney's post-hoc tests showed statistically significant differences in CGRP expression between two pulpitis groups (P < 0.05), differences in SP expression between symptomatic and asymptomatic irreversible pulpitis groups were not significant. This study demonstrated that the expression of CGRP and SP is significantly higher in pulps with irreversible pulpitis compared with healthy pulps. [source] Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variantsBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2010T Benned-Jensen Background and purpose:, In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms. Experimental approach:, Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [35S]-GTP,S binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC4 and LTD4. Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively. Key results:, The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC50. Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through G,i. Conclusions and implications:, We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions. [source] | ||||||||||