interacts_with DB00243?
5-Azacitidine restores and amplifies the bicalutamide response on preclinical models of androgen receptor expressing or deficient prostate tumors . BACKGROUND : Epigenetic modifications play a key role in the in prostate cancer ( Pca ) progression to a hormone refractory state ( HRPC ) and the current use of agents targeting epigenetic changes has become a topic of intense interest in cancer research . In this regard , 5-Azacitine ( 5-Aza ) represents a promising epigenetic modulator . This study tested the hypothesis that 5-Aza may restore and enhance the responsiveness of HRPC cells to anti-hormonal therapy on P10275 ( AR ) expressing ( 22rv1 ) and AR-deficient ( PC3 ) cells . METHODS : The effects were studied in vitro and in vivo models . This sequential treatment induced in vitro cell cycle arrest and apoptosis both in 22rv1 and PC3 tumor cell lines . RESULTS : This combined treatment up-regulated the expression of P48023 , phospho- Q13158 , p16(INKA) , Bax , Bak , and P38936 ( P38936 ) , and inhibited FLIP , Bcl-2 , and Bcl-XL expression . The re-activation of hormonal response of AR-negative PC3 cell line was partially due to the AR re-expression mediated by 5-Aza treatment . In contrast , the increase in the response to anti-androgenic therapy in 22rv1 did not correlate with AR expression levels . Furthermore , xenograft studies revealed that the combined treatment of 5-Aza with AR-antagonist DB01128 had additive/synergistic effects in repressing tumor growth in vivo and the underlying mechanisms responsible for these effects seem to be in part mediated by induction of apoptosis . CONCLUSIONS : So , this study strongly suggests a therapeutic potential of 5-Aza in combination with anti-androgen therapy in patients with in AR expressing and AR-deficient HRPC . Functional characterization of a novel serotonin receptor ( 5-HTap2 ) expressed in the CNS of Aplysia californica . Serotonin has been shown to be a neuromodulator in the Aplysia californica CNS . The diversity of serotonin actions is due to the existence of several different receptor subtypes . In this study we report the cloning of a full-length cDNA , coding for a novel serotonin receptor ( 5-HTap2 ) . The receptor protein bears the characteristics of G protein-coupled receptors . It shares 68 % and 34 % of its amino acid sequence identity with the 5-HTlym receptor from Lymnaea stagnalis and the mammalian P08908 receptor , respectively . When transfected in P29320 293 cells , 5-HTap2 was negatively coupled to adenylate cyclase . Ligand binding analysis indicated that the order of potencies of various drugs for the inhibition of [3H]LSD binding was : methiothepin > metergoline > 5-CT > PAPP > 5-HT > ketanserin > NAN-190 > 8-OH-DPAT > clozapine . RT-PCR amplification of RNA isolated from different tissues indicated that this receptor is expressed in the CNS and in bag cells . The expression of 5-HTap2 restricted to the CNS suggests an important role for this receptor in the modulation of neuronal functions in Aplysia . Moreover , the high expression of 5-HTap2 in the bag cells , associated with its pharmacological profile , suggests that this receptor may be implicated in modulating the afterdischarge during the egg-laying behavior . Comparison of the novel antipsychotic ziprasidone with clozapine and olanzapine : inhibition of dorsal raphe cell firing and the role of P08908 receptor activation . Ziprasidone is a novel antipsychotic agent which binds with high affinity to P08908 receptors ( Ki = 3.4 nM ) , in addition to P28221 , 5-HT2 , and D2 sites . While it is an antagonist at these latter receptors , ziprasidone behaves as a P08908 agonist in vitro in adenylate cyclase measurements . The goal of the present study was to examine the P08908 properties of ziprasidone in vivo using as a marker of central P08908 activity the inhibition of firing of serotonin-containing neurons in the dorsal raphe nucleus . In anesthetized rats , ziprasidone dose-dependently slowed raphe unit activity ( ED50 = 300 micrograms/kg i.v. ) as did the atypical antipsychotics clozapine ( ED50 = 250 micrograms/kg i.v. ) and olanzapine ( ED50 = 1000 micrograms/kg i.v. ) . Pretreatment with the P08908 antagonist WAY-100,635 ( 10 micrograms/kg i.v. ) prevented the ziprasidone-induced inhibition ; the same dose of WAY-100,635 had little effect on the inhibition produced by clozapine and olanzapine . Because all three agents also bind to alpha 1 receptors , antagonists of which inhibit serotonin neuronal firing , this aspect of their pharmacology was assessed with desipramine ( DB01151 ) , a NE re-uptake blocker previously shown to reverse the effects of alpha 1 antagonists on raphe unit activity . DB01151 ( 5 mg/kg i.v. ) failed to reverse the inhibitory effect of ziprasidone but produced nearly complete reversal of that of clozapine and olanzapine . These profiles suggest a mechanism of action for each agent , P08908 agonism for ziprasidone and alpha 1 antagonism for clozapine and olanzapine . The P08908 agonist activity reported here clearly distinguishes ziprasidone from currently available antipsychotic agents and suggests that this property may play a significant role in its pharmacologic actions . Constitutive high-level production of human lymphotoxin by CHO- P04264 cells transformed with the human lymphotoxin gene controlled by a human beta-actin promoter . To achieve high-level production of human lymphotoxin ( hLT ) , a plasmid ( p beta LT-ldhfr ) containing the hLT genomic DNA , a mouse dihydrofolate reductase ( P00374 ) cDNA , and a bacterial Ecogpt gene was cotransfected with a plasmid ( p beta LTML ) encoding only the hLT genomic DNA into Chinese hamster ovary ( CHO- P04264 ) cells at a 1:7 molar ratio . Subsequently one of the Ecogpt-positive clones ( clone A31 ) was grown in stepwise increasing concentrations of methotrexate ( MTX ) . A large amount of the hLT was secreted by cells resistant to increased levels of MTX as a result of coamplification of the P00374 cDNA and the hLT gene . A cell clone ( clone M-1 ) resistant to up to 500 nM MTX constitutively expressed the hLT at a concentration of 80 micrograms per ml at an elevated level for about 2 months . The hLT was produced in glycosylated form the molecular mass of which was 23,000 daltons and the mRNA was normally spliced , so the protein molecules were probably homogeneous . Flow cytometric analysis of mammalian glial cultures treated with methotrexate . DB00563 ( MTX ) is an antineoplastic drug that acts by competitive inhibition of the enzyme dihydrofolate reductase ( P00374 ) . MTX treatment of cultured cell lines leads to the emergence of resistant cell populations . Studies using stepwise selection procedures have demonstrated that MTX resistance conferred by overproduction of P00374 can be caused by P00374 gene amplification . We examined the effect of MTX on cells whose origin more closely approximates the in vivo condition by developing a culture system using dissociated brain tissue from 17-19 day old mouse embryos . At the first passage , cultures were divided into control and MTX groups . Cells were treated with the same or successively higher concentrations of MTX at each passage over a 3-4 month period . The first passage eliminated neurons and left a glial culture comprised of approximately 90 % astrocytes . We used the Fluorescence Activated Cell Sorter in conjunction with fluorescent dyes to measure P00374 content , DNA content , size , and viability of glial cells following MTX treatment . MTX-treated cells divided but grew more slowly and were larger than untreated cells . Stepwise selection in 30/60/90 nM or 60/120 nM MTX resulted in significant two- to threefold increases in fluorescence , and hence P00374 levels . Slot hybridizations assays demonstrated a threefold increase in P00374 gene copy number in the DNA from the 30/60/90 cultures . Thus , our findings were consistent with the results obtained from somatic cell lines , and lend support to the hypothesis that gene amplification may be a common mechanism for the acquisition of resistance in many types of cells . They also indicate that glial cells may be a specific target for cytotoxic effects of MTX on the central nervous system . beta-Arrestin-2 interaction and internalization of the human P47900 receptor are dependent on C-terminal phosphorylation sites . The nucleotide receptor P2Y(1) regulates a variety of physiological processes and is involved in platelet aggregation . Using human P2Y(1)-receptors C-terminally fused with a fluorescent protein , we studied the role of potential receptor phosphorylation sites in receptor internalization and beta-arrestin-2 translocation by means of confocal microscopy . Three receptor constructs were generated that lacked potential phosphorylation sites in the third intracellular loop , the proximal C terminus , or the distal C terminus . The corresponding receptor constructs were expressed in human embryonic kidney ( P29320 ) -293 cells and stimulated with 100 muM ADP . Rapid receptor internalization was observed for the wild-type receptor and from those constructs mutated in the third intracellular loop and the proximal C terminus . However , the construct lacking phosphorylation sites at the distal C terminus did not show receptor internalization upon stimulation . The microscopic data were validated by HA-tagged receptor constructs using a cell surface enzyme-linked immunosorbent assay . P2Y(1)-receptor stimulated beta-arrestin-2-yellow fluorescent protein ( YFP ) translocation followed the same pattern as receptor internalization . Hence , no beta-arrestin-2-YFP translocation was observed when the distal C-terminal phosphorylation sites were mutated . Individual mutations indicate that residues Ser352 and Thr358 are essential for receptor internalization and beta-arrestin-2-YFP translocation . In contrast , protein kinase C ( PKC ) -mediated receptor desensitization was not affected by mutation of potential phosphorylation sites in the distal C terminus but was prevented by mutation of potential phosphorylation sites in the proximal C terminus . P2Y(1)-receptor internalization in P29320 -293 cells was not blocked by inhibitors of PKC and calmodulin-dependent protein kinase . Thus , we conclude that P2Y(1)-receptor desensitization and internalization are mediated by different phosphorylation sites and kinases . The expression of retinoblastoma and Sp1 is increased by low concentrations of cyclin-dependent kinase inhibitors . We examined the effect of suboptimal concentrations of cyclin-dependent kinase inhibitors , which do not interfere with cell proliferation , on retinoblastoma expression in hamster ( Chinese hamster ovary P04264 ) and human ( K562 and HeLa ) cells . To achieve this , we used the chemical inhibitors roscovitine and olomoucine ( which inhibit P24941 preferentially ) , P55089 -01 ( which also inhibits P11802 /6 ) and P38936 ( as an intrinsic inhibitor ) . All chemical inhibitors and overexpression of P38936 strongly induced retinoblastoma protein expression . P55089 -01-mediated retinoblastoma expression was caused by an increase in both the levels of retinoblastoma mRNA and the stability of the protein . The expression of the transcription factor Sp1 , a retinoblastoma-interacting protein , was also enhanced by all the cyclin-dependent kinase inhibitors tested . However , Sp1 expression was caused by an increase in the levels of Sp1 mRNA without modification in the stability of the protein . By using luciferase experiments , the transcriptional activation of both retinoblastoma and Sp1 promoters by P55089 -01 was confirmed . Bisindolylmaleimide I , at concentrations causing a similar or higher inhibition of protein kinase C than P55089 -01 , provoked a lower activation of retinoblastoma and Sp1 expression . Finally , the effects of cyclin-dependent kinase inhibitors on dihydrofolate reductase gene expression were evaluated . Treatment with P55089 -01 increased cellular dihydrofolate reductase mRNA levels , and dihydrofolate reductase enzymatic activity was enhanced by P55089 -01 , roscovitine , olomoucine and P38936 , in transient transfection experiments . These results support a mechanism for the self-regulation of retinoblastoma expression , and point to the need to establish the appropriate dose of cyclin-dependent kinase inhibitors as antiproliferative agents in anticancer treatments . Is phentermine an inhibitor of monoamine oxidase ? A critical appraisal . DB00191 produces a spectrum of concentration-dependent biochemical effects . It interacts with NE transporters at 0.1 microM , DA transporters at about 1 microM , 5-HT transporters at 15 microM and P21397 at about 100 microM . When administered at typical anorectic doses , phentermine primarily interacts with DA and NE transporters and does not produce biochemical or neurochemical effects which would occur if it were inhibiting P21397 . Some other explanation other than MAO inhibition must be sought to explain how oral phentermine increases platelet 5-HT , since platelet P27338 does not metabolize platelet 5-HT , and since amphetamine-type drugs are even weaker inhibitors of P27338 than P21397 . Clinical studies in humans have shown that amphetamine , which is a more potent inhibitor of P21397 than phentermine , does not inhibit P21397 at therapeutic doses . Neither phentermine alone , fluoxetine alone or their combined use have been associated with cardiac valvulopathy , and clinical experience has shown their combined use to be free of significant adverse effects . Viewed collectively , there appears to be no data to support the hypothesis that phentermine inhibits MAO at typical therapeutic doses . DB00203 inhibits calcineurin/ Q13469 -mediated cyclin A expression in pulmonary artery smooth muscle cells . AIMS : To examine whether calcineurin/NFAT signaling pathway leads to proliferation of pulmonary artery smooth muscle cells ( PASMCs ) by regulating cell cycle proteins and whether the phosphodiesterase-5 ( O76074 ) inhibitor sildenafil affects calcineurin/NFAT-induced cell proliferation . MAIN METHODS : A [(3)H]thymidine incorporation assay was used to examine DNA synthesis ( cell proliferation ) ; cyclin A and Q13469 expressions were determined by Western blot . P24941 ( P24941 ) activity was measured with an in vitro kinase activity assay , and calcineurin and NFAT activity were evaluated using a calcineurin assay kit and a luciferase activity assay , respectively . A chemical inhibitor or siRNA transfection was used to inhibit calcineurin/NFAT signaling pathway . KEY FINDINGS : Serotonin dose-dependently stimulated cyclin A expression in PASMCs . This effect was accompanied by dose-dependent increases in P24941 activity and the rate of DNA synthesis . At the same time , PASMCs treated with serotonin showed dose-dependent activation of calcineurin/NFAT signaling pathway . Inhibition of calcineurin activity by cyclosporine A or loss of Q13469 protein by siRNA transfection abolished serotonin-induced cyclin A expression and consequent P24941 activation and DNA synthesis . We further found that pretreatment of cells with sildenafil suppressed serotonin-triggered activation of calcineurin/ Q13469 signaling pathway and resultant cyclin A expression , P24941 activation and cell proliferation , while the presence of DT-3 [ a specific protein kinase G ( PKG ) peptide inhibitor ] reversed the effects of sildenafil on PASMCs . SIGNIFICANCE : Our study suggests that enhanced PKG activity suppresses calcineurin/ Q13469 cascade-mediated cyclin A expression , P24941 activation and PASMC proliferation to contribute to the overall effects of sildenafil in the treatment of pulmonary hypertension . Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction . A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells . We have used the baculovirus transduction system , BacMam , to demonstrate transient expression of multi-subunit KATP channels in CHO- P04264 and P29320 -293 EBNA cells using sulfonylurea receptor 1 ( Q09428 ) , SUR2A , SUR2B , and P55040 6.2 genes . [ 3H ] -glyburide binding , patch clamp , and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression . BacMam delivery of each Q09428 isoform with KIR6.2 was demonstrated based on its pharmacological profiles . Expression levels of Q09428 and KIR6.2 were titrated by varying the viral concentration or time of virus addition , with functional activity measured in as little as 4-6 hours posttransduction . Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel . Independently altering treatment with virus containing either the Q09428 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane . This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function . [ Prominent features of management strategies in acute coronary syndromes with the new oral antiplatelet agents ] . The novel oral Q9H244 inhibitors ( prasugrel and ticagrelor ) have been incorporated into the recently updated acute coronary syndrome ( ACS ) guidelines , as an adjunct antiplatelet treatment to aspirin . The studies involving the use of new oral antiplatelet agents that are more potent , predictable and faster platelet inhibitors than clopidogrel have demonstrated superiority with respect to the primary composite endpoint ( cardiovascular death , non-lethal myocardial infarction , stroke ) for both prasugrel and ticagrelor compared to clopidogrel . The subgroup analysis of the relevant studies showed that these new agents differ in their level of efficacy in different ACS patient subgroups : ( 1 ) Mortality was reduced with ticagrelor ; ( 2 ) DB08816 is especially more effective in intermediate-and high-risk non-ST elevation ACS patients in whom early invasive strategy is selected ; ( 3 ) Prasugrel should be especially preferred in patients with acute ST elevation myocardial infarction undergoing percutaneous coronary intervention ( P05154 ) after diagnostic angiography ; and ( 4 ) Prasugrel is more effective in diabetic patients . While clopidogrel is recommended for ACS patients who are followed with a non-invasive strategy or who have not undergone percutaneous revascularization , it is the last line choice or an alternative to the Q9H244 inhibitor therapy for patients undergoing invasive strategy . P2Y receptor antagonists in thrombosis . The dual role of P47900 and Q9H244 receptors in platelet aggregation by ADP has been firmly established , based on the action of selective inhibitors , gene targeting in mice and human genetic evidence . Both of these receptor subtypes constitute targets for antithrombotic agents , and compounds with a dual action might also be of interest . However , the agents currently on the market ( ticlopidine and clopidogrel ) , or known to be in development ( cangrelor , DB08816 and prasugrel ) , all target the Q9H244 receptor . The thienopyridines ( ticlopidine , clopidogrel and prasugrel ) irreversibly inactivate the Q9H244 receptor via the covalent binding of an active metabolite generated in the liver , while the other compounds are competitive antagonists . DB06441 , an DB00171 derivative , is suitable for intravenous perfusion , whereas DB08816 is in clinical development as an orally active agent . Agonist-promoted down-regulation and functional desensitization in two naturally occurring variants of the human serotonin1A receptor . We recently reported two naturally occurring polymorphisms of the human serotonin1A ( P08908 ) receptor : glycine22 --> serine ( Ser22 ) and isoleucine28 --> valine ( Val28 ) in the putative aminoterminal domain of the receptor . To investigate the regulatory properties of these variants , the wild type ( WT ) and variant P08908 receptors were stably expressed in CHO- P04264 cells . WT , Ser22 , and Val28 displayed similar high-affinity binding to [ 3H ] -8-OH-DPAT . Competition experiments with P08908 agonists and antagonists demonstrated similar pharmacological profiles . Receptor agonist-promoted down-regulation was tested by exposure to 100 mumol/L 8-OH-DPAT . After 24-h exposure , WT and Val28 underwent 59.3 +/- 3.9 % and 59.5 +/- 1.4 % reduction in receptor density respectively , whereas the degree of down-regulation was significantly lower for Ser22 ( 21.4 +/- 4.2 % ) . Cell treatment for 24 h with 100 mumol/L 8-OH-DPAT reduced the 5-HT-induced inhibition of DB02527 accumulation by 24.9 +/- 5.1 % for WT and 16.4 +/- 0.8 % for Val28 , but only by 4.8 +/- 3 % for Ser22 . We conclude that the Ser22 variant is capable of attenuating agonist-mediated receptor down-regulation and desensitization . P01375 -related apoptosis inducing ligand ( P50591 ) up-regulates death receptor 5 ( DR5 ) mediated by NFkappaB activation in epithelial derived cell lines . P01375 -related apoptosis inducing ligand ( P50591 /APO-2L ) activates nuclear factor kappaB ( NFkappaB ) . This activation is regulated by the recruitment of an adaptor protein Fas associating death domain ( Q13158 ) to P50591 death receptors , death receptor 4 ( DR4 , O00220 ) and death receptor 5 ( DR5 O14763 ) . This leads to recruitment of caspase 8 and receptor interacting protein ( RIP ) to the receptor complex . Upon recruitment of caspase 8 and RIP , NFkappaB inducing kinase ( NIK ) becomes activated causing NFkappaB activation . The role of P50591 induced NFkappaB activation in epithelial cells is unknown . Herein we demonstrate that P50591 increases expression of DR5 in human embryonic kidney ( P29320 ) 293 , MCF-7 and MDA MB 231 epithelial cell lines while DR4 expression remains unchanged . Blockage of NFkappaB activation either by expression of dominant negative IkappaB or treatment with proteasome inhibitor lactacystin eliminates P50591 induced DR5 expression . Expression of Q13158 dominant negative in P29320 293 cells that prevents the recruitment of caspase 8 and RIP to P50591 death receptors also eliminates this increase . By over expression of the p65 subunit of NFkappaB that increases NFkappaB transcriptional activity , DR5 expression was increased compared to vector alone expressing cells . By blocking P50591 induced NFkappaB activation , the sensitivity of cells to undergo P50591 induced apoptosis was significantly decreased . Conversely , the amount of P50591 induced apoptosis was increased in P29320 293 cells over expressing p65 subunit of NFkappaB . Finally blockage of NFkappaB activation eliminates the synergistic apoptotic response of P50591 and etoposide . Thus , P50591 mediated NFkappaB activation increases DR5 expression thereby amplifying the apoptotic response of P50591 in epithelial derived cells . Synthesis and evaluation of ( S ) -2-(2-[18F]fluoroethoxy)-4- ( [ 3-methyl-1-(2-piperidin-1-yl-phenyl)-butyl-carbamoyl ] -methyl ) -benzoic acid ( [18F]repaglinide ) : a promising radioligand for quantification of pancreatic beta-cell mass with positron emission tomography ( PET ) . 18F-labeled non-sulfonylurea hypoglycemic agent ( S ) -2-(2-[(18)F]fluoroethoxy)-4- ( ( 3-methyl-1-(2-piperidin-1-yl-phenyl)-butylcarbamoyl ) -methyl ) -benzoic acid ( [(18)F]repaglinide ) , a derivative of the sulfonylurea-receptor ( Q09428 ) ligand repaglinide , was synthesized as a potential tracer for the non-invasive investigation of the sulfonylurea 1 receptor status of pancreatic beta-cells by positron emission tomography ( PET ) in the context of type 1 and type 2 diabetes . [(18)F] DB00912 could be obtained in an overall radiochemical yield ( RCY ) of 20 % after 135 min with a radiochemical purity higher than 98 % applying the secondary labeling precursor 2-[(18)F]fluoroethyltosylate . Specific activity was in the range of 50-60 GBq/micromol . Labeling was conducted by exchanging the ethoxy-moiety into a 2-[(18)F]fluoroethoxy group . To characterize the properties of fluorinated repaglinide , the affinity of the analogous non-radioactive (19)F-compound for binding to the human Q09428 isoform was assessed . [(19)F] DB00912 induced a complete monophasic inhibition curve with a Hill coefficient close to 1 ( 1.03 ) yielding a dissociation constant ( K(D) ) of 134 nM . Biological activity was proven via insulin secretion experiments on isolated rat islets and was comparable to that of repaglinide . Finally , biodistribution of [(18)F]repaglinide was investigated in rats by measuring the concentration of the compound in different organs after i.v. injection . Pancreatic tissue displayed a stable accumulation of approximately 0.12 % of the injected dose from 10 min to 30 min p.i . 50 % of the radioactive tracer could be displaced by additional injection of unlabeled repaglinide , indicating that [(18)F]repaglinide might be suitable for in vivo investigation with PET . Mechanisms of atrial-selective block of Na⁺ channels by ranolazine : I . Experimental analysis of the use-dependent block . Atrial-selective inhibition of cardiac Na(+) channel current ( I(Na) ) and I(Na)-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation . The present study examined the basis for the atrial-selective actions of ranolazine . Whole cell I(Na) was recorded at 15°C in canine atrial and ventricular myocytes and in human embryonic kidney ( P29320 ) -293 cells expressing Q14524 . Tonic block was negligible at holding potentials from -140 to -100 mV , suggesting minimal drug interactions with the closed state . Trains of 40 pulses were elicited over a range of holding potentials to determine use-dependent block . Guarded receptor formalism was used to analyze the development of block during pulse trains . Use-dependent block by ranolazine increased at more depolarized holding potentials , consistent with an interaction of the drug with either preopen or inactivated states , but was unaffected by longer pulse durations between 5 and 200 ms , suggesting a weak interaction with the inactivated state . Block was significantly increased at shorter diastolic intervals between 20 and 200 ms . Responses in atrial and ventricular myocytes and in P29320 -293 cells displayed a similar pattern . DB00243 is an open state blocker that unbinds from closed Na(+) channels unusually fast but is trapped in the inactivated state . Kinetic rates of ranolazine interactions with different states of atrial and ventricular Na(+) channels were similar . Our data suggest that the atrial selectivity of ranolazine is due to a more negative steady-state inactivation curve , less negative resting membrane potential , and shorter diastolic intervals in atrial cells compared with ventricular cells at rapid rates . Development of stable cell lines expressing different subtypes of GABAA receptors . The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors . For this , we developed an original two step selection strategy , in which the first step consisted of transfecting P29320 293 cells with rat GABAA receptor alpha and beta subunits . G 418 resistant colonies isolated at this step were screened for [ 3H ] muscimol binding to select for those that coexpressed alpha- and beta-subunits . The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant P00374 gene . After a second round of selection , this time in presence of methotrexate , those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [ 3H ] flumazenil as a probe . This strategy was applied to the isolation of 3 GABAA receptor clones , alpha 1 beta 2 gamma 2s , alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s , that expressed relatively high levels of these proteins . These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations . These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes . Intake of vitamin P04264 and K2 and risk of hip fractures : The Hordaland Health Study . BACKGROUND : Evidence of the effect of vitamin K on bone health is conflicting . The aim was to investigate the association between intake of vitamins P04264 and K2 and subsequent risk of hip fracture in a general population sample , as well as potential effect modification by apolipoprotein E gene ( P02649 ) status by presence of the E4 allele . METHODS : 1238 men and 1569 women 71-75 years of age were included in the community-based Hordaland Health Study 1997-1999 in Western Norway . Information on hip fracture was obtained from hospitalizations in the region from enrolment until 31 December 2009 . Information on intake of vitamins P04264 and K2 collected at baseline was used as potential predictors of hip fracture in Cox proportional hazards regression analyses . RESULTS : Participants in the lowest compared to the highest quartile of vitamin P04264 intake had increased risk of suffering a hip fracture ( hazard ratio (HR)=1.57 [ 95 % CI 1.09 , 2.26 ] ) . Vitamin K2 intake was not associated with hip fracture . Presence of APOE4-allele did not increase the risk of hip fracture , nor was there any effect modification with vitamin P04264 in relation to risk of hip fracture . CONCLUSIONS : A low intake of vitamin P04264 , but not K2 , was associated with an increased risk of hip fractures . [ Functional analysis of the alpha1G subunit of the T-type calcium channel in cellular proliferation ] . Increases of functional T-type calcium channel ( T-channel ) expression have been associated with cellular proliferation although evidence for this remains controversial . In the present study , we have used a variety of cellular , molecular and electrophysiological techniques to test the hypothesis that T-type channels play a causal role in the signaling pathway leading to proliferation . The results showed that stable over-expression of alpha1G T-channel subunit in P29320 -293 cells conferred a significant growth advantage . Thus , cell population doubling time was reduced to 13.7 +/- 0.3 h in alpha1G transfectants , compared to control cultures ( 22.1 +/- 1.1 h ) and flow cytometry analysis showed that this was due to a reduction in the number of alpha1G transfectants residing in the G0/ P55008 phases of the cell cycle compared to controls . The selective T-type calcium channel blocker , mibefradil , induced a dose-dependent inhibition of proliferation in alpha1G tansfectants . Furthermore , the Western blotting results proved that the level of protein expression of P24941 , cyclin A and cyclin E was high in alpha1G transfectants compared to control cultures . Our results demonstrate that the T-type calcium channel provides a significant growth advantage to P29320 -293 cells that might occur via effects on the P55008 /S cell cycle mechanism . [ Construction of mammalian cell expression vector for pAcGFP-bFADD fusion protein and its expression in CHO- P04264 cell ] . Fas-associated death domain ( Q13158 ) is a signal connection protein in Fas/ P48023 apoptotic path which might play a key role on apoptosis by transferring apoptotic signal . To reveal the intracellular signal transduction molecules involved in the procedure of follicular development in bovine ovary , we cloned Q13158 gene in bovine ovary tissue with RT-PCR , deleted the termination codon in its cDNA and directionally cloned the amplified Q13158 gene into eukaryotic expression vector pAcGFP-N1 including AcGFP , successfully constructed the fusion protein recombinant plasmid . After identifying by restrictive enzyme Bgl II/EcoR I and sequencing , transfected pAcGFP-bFADD into CHO- P04264 cell mediated by Lipofectamine 2000 , observed the expression of AcGFP and detected the transcription and expression of Q13158 by RT-PCR and Western blotting . The results showed that the cattle Q13158 was successfully cloned , the pAcGFP-bFADD fusion protein recombinant plasmid was successfully constructed by introducing Bgl II , EcoR I cloning site at two ends of Q13158 open reading frame and inserting a Kozak sequence before start codon . AcGFP expression was detected as early as 24 h after transfection . The percentage of AcGFP positive cells reached about 65 % after 24 h . A 654 bp transcription was amplified by RT-PCR , and 51.4 kD target protein was detected by Western blotting . Construction of pAcGFP-bFADD recombinant plasmid should be helpful for further understanding the mechanism of regulation of Q13158 on bovine oocytes formation and development . Potent and selective activation of the pancreatic beta-cell type K( DB00171 ) channel by two novel diazoxide analogues . AIMS/HYPOTHESIS : We investigated the pharmacological properties of two novel DB00171 sensitive potassium ( K( DB00171 ) ) channel openers , 6-Chloro-3-isopropylamino-4 H-thieno [ 3,2- e ] -1,2,4-thiadiazine 1,1-dioxide ( NNC 55-0118 ) and 6-chloro-3-(1-methylcyclopropyl)amino-4 H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide ( NN414 ) , on the cloned cardiac ( Kir6.2/SUR2A ) , smooth muscle ( Kir6.2/SUR2B ) and pancreatic beta cell ( Kir6.2/ Q09428 ) types of K( DB00171 ) channel . METHODS : We studied the effects of these compounds on whole-cell currents through cloned K( DB00171 ) channels expressed in Xenopus oocytes or mammalian cells ( HEK293 ) . We also used inside-out macropatches excised from Xenopus oocytes . RESULTS : In P29320 293 cells , NNC 55-0118 and NN414 activated Kir6.2/ Q09428 currents with EC(50) values of 0.33 micromol/l and 0.45 micromol/l , respectively , compared with that of 31 micro mol/l for diazoxide . Neither compound activated Kir6.2/SUR2A or Kir6.2/SUR2B channels expressed in oocytes , nor did they activate Kir6.2 expressed in the absence of Q09428 . Current activation was dependent on the presence of intracellular MgATP , but was not supported by MgADP . CONCLUSION/INTERPRETATION : Both NNC 55-0118 and NN414 selectively stimulate the pancreatic beta-cell type of K( DB00171 ) channel with a higher potency than diazoxide , by interaction with the Q09428 subunit . The high selectivity and efficacy of the compounds could prove useful for treatment of disease states where inhibition of insulin secretion is beneficial . Synthesis of new thiazolo[4,5-d]pyrimidines as DB01285 releasing factor modulators . P06850 ( CRF ) is a neurohormone that plays a crucial role in integrating the body 's overall response to stress . It appears necessary and sufficient for the organism to mount functional , physiological and endocrine responses to stressors . CRF is released in response to various triggers such as chronic stress . The role of CRF and its involvement in these neurological disorders suggest that new drugs that can target the CRF function or bind to its receptors may represent a new development of neuropsychiatric medicines to treat various stress-related disorders including depression , anxiety and addictive disorders . Based on pharmacophore of the CRF1 receptor antagonists , a new series of thiazolo[4,5-d] pyrimidines were synthesized as P06850 ( CRF ) receptor modulators and the prepared compounds carry groups shown to produce optimum binding affinity to CRF receptors . Twenty two compounds were evaluated for their CRF1 receptor binding affinity in P29320 293 cell lines and two compounds 5o and 5s showed approximately 25 % binding affinity to CRF1 receptors . Selected compounds ( 5c and 5f ) were also evaluated for their effect on expression of genes associated with depression and anxiety disorders such as CRF1 , P16220 , P21397 , P31645 , P01303 , DatSLC6a3 , and P09172 and significant upregulation of CRF1 mRNA has been observed with compound 5c . Identification of a potent inverse agonist at a constitutively active mutant of human Q9H244 receptor . Human platelets express two P2Y receptors : G(q)-coupled P2Y(1) , and G(i)-coupled P2Y(12) . Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis . Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems , but in the P2Y receptors , to date , no constitutive activity has been reported . In our effort to identify G protein coupling domains of the human platelet ADP receptor , we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary- P04264 cells . It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity , as evidenced by decreased DB02527 levels in the absence of agonists . The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin , a G(i) inhibitor . The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP , with an EC(50) of 0.15 +/- 0.04 nM . The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin . Pharmacological evaluation of several P2Y(12) antagonists revealed ( E ) -N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid ( AR-C78511 ) as a potent P2Y(12) inverse agonist and 5'-adenylic acid , N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]- , monoanhydride with (dichloromethylene)bis [ phosphonic acid ] ( AR-C69931MX ) as a neutral antagonist . In conclusion , this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist . Multiple arrhythmic syndromes in a newborn , owing to a novel mutation in Q14524 . BACKGROUND : Mutations in the Q14524 gene have been linked to Brugada syndrome ( BrS ) , conduction disease , Long QT syndrome ( LQT3 ) , atrial fibrillation ( AF ) , and to pre- and neonatal ventricular arrhythmias . OBJECTIVE : The objective of this study is to characterize a novel mutation in Na(v)1.5 found in a newborn with fetal chaotic atrial tachycardia , post-partum intraventricular conduction delay , and QT interval prolongation . METHODS : Genomic DNA was isolated and all exons and intron borders of 15 ion-channel genes were sequenced , revealing a novel missense mutation ( Q270K ) in Q14524 . Na(v)1.5 wild type ( WT ) and Q270K were expressed in CHO- P04264 with and without the Na(v)β1 subunit . Results . Patch-clamp analysis showed ∼40 % reduction in peak sodium channel current ( I(Na) ) density for Q270K compared with WT . Fast and slow decay of I(Na) were significantly slower in Q270K . Steady-state activation and inactivation of Q270K channels were shifted to positive potentials , and window current was increased . The tetrodotoxin-sensitive late I(Na) was increased almost 3-fold compared with WT channels . DB00243 reduced late I(Na) in WT and Q270K channels , while exerting minimal effects on peak I(Na) . CONCLUSION : The Q270K mutation in Q14524 reduces peak I(Na) while augmenting late I(Na) , and may thus underlie the development of atrial tachycardia , intraventricular conduction delay , and QT interval prolongation in an infant . Genetic factors influencing outcome from neurotrauma . PURPOSE OF REVIEW : Clinical outcome after neurotrauma is considerably variable and can only partly be explained by known prognostic factors . There is converging evidence from genetic research that a number of genetic variants may contribute to this variability . This review provides recent data from human studies , published in the previous year , on genetic factors influencing outcome after neurotrauma . The bibliographic databases MEDLINE , EMBASE and PsycINFO were searched to identify relevant studies . RECENT FINDINGS : Genetic susceptibility to various aspects of clinical outcome after neurotrauma was reported in recent clinical studies . Genetic loci investigated include polymorphisms in P02649 , P21397 , P23560 , NOS3 , P05231 , P12036 , P31645 , P21964 , P48454 and Q8IX03 genes . The importance of these findings and future directions are discussed . SUMMARY : Recent genetic studies have revealed emerging aspects and extended the existing knowledge regarding the pathogenesis of neurotrauma and the genetic influence on phenotypic diversity . A better understanding of the underlying biological pathways and molecular mechanisms of an individual 's response to neurotrauma may hold the promise of novel treatment strategies and improved clinical outcome .