interacts_with DB00243?
Can a cocktail designed for phenotyping pharmacokinetics and metabolism enzymes in human be used efficiently in rat ? We recently designed the CIME cocktail consisting of 10 drugs to assess the activity of the major human CYPs ( P05177 , P10632 , P11712 , P33261 , P10635 and CYP3A ) , a phase II enzyme ( P22309 /6/9 ) , two drug transporters ( P-gp and Q9Y6L6 ) and a component of the renal function ( Videau et al. 2010 ) . The present work aimed at studying the usefulness of the CIME cocktail in the rat.The CIME cocktail was given per os to three male and three female rats , or incubated with rat liver microsomes . Parent substrates and metabolites were quantified by LC-MS/MS in plasma , urine and hepatic microsomal media , and phenotyping index were subsequently calculated.The CIME cocktail could therefore be used in the rat to phenotype rapidly and simultaneously CYP3A1/2 with omeprazole/omeprazole-sulfone , midazolam/1'-hydroxymidazolam or 4-hydroxymidazolam and/or dextromethorphan/3-methoxymorphinan , CYP2C6/11 with tolbutamide/4-hydroxytolbutamide , CYP2D1/2 with omeprazole/5-hydroxyomeprazole or dextromethorphan/dextrorphan , and P19224 /7 with acetaminophen/acetaminophen-glucuronide . Our results confirmed also several known gender differences and brought new information on the urinary excretion of rosuvastatin . However , the major rat CYPs , CYP2C11 and CYP2C12 , are not specifically assessed . An optimized version of the CIME cocktail should therefore be designed and would be of major importance to more largely phenotype Q09013 enzymes in rats to study Q09013 variability factors such as disease , age , or to exposure to inductors or inhibitors . Targeting Q01196 / Q06455 -histone deacetylase repressor complex : a novel mechanism for valproic acid-mediated gene expression and cellular differentiation in Q01196 / Q06455 -positive acute myeloid leukemia cells . In t(8;21) acute myeloid leukemia ( AML ) , the Q01196 / Q06455 fusion protein promotes leukemogenesis by recruiting class I histone deacetylase ( HDAC ) -containing repressor complex to the promoter of Q01196 target genes . Valproic acid ( DB00313 ) , a commonly used antiseizure and mood stabilizer drug , has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition . DB00313 causes selective proteasomal degradation of Q92769 but not other class I HDACs ( i.e. , HDAC 1 , 3 , and 8 ) . Therefore , we raised the question of whether this drug can effectively target the leukemogenic activity of the Q01196 / Q06455 fusion protein that also recruits Q13547 , a key regulator of normal and aberrant histone acetylation . We report here that DB00313 treatment disrupts the Q01196 / Q06455 - Q13547 physical interaction , stimulates the global dissociation of Q01196 / Q06455 - Q13547 complex from the promoter of Q01196 / Q06455 target genes , and induces relocation of both Q01196 / Q06455 and Q13547 protein from nuclear to perinuclear region . Furthermore , we show that mechanistically these effects associate with a significant inhibition of HDAC activity , histone H3 and H4 hyperacetylation , and recruitment of RNA polymerase II , leading to transcriptional reactivation of target genes ( i.e. , P08700 ) otherwise silenced by Q01196 / Q06455 fusion protein . Ultimately , these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis . Taken together , these data support the notion that DB00313 might effectively target Q01196 / Q06455 -driven leukemogenesis through disruption of aberrant Q13547 function and that DB00313 should be integrated in novel therapeutic approaches for Q01196 / Q06455 -positive AML . P35372 and P20813 gene variants as risk factors in methadone-related deaths . DB00333 is a medication valued for its effectiveness in the treatment of heroin addiction ; however , many fatal poisonings associated with its use have been reported over the years . We have examined the association between P20813 and micro-opioid receptor ( P35372 ) gene variations and apparent susceptibility to methadone poisoning . Genomic DNA was extracted from postmortem whole blood of 40 individuals whose deaths were attributed to methadone poisoning . The presence of P20813 *4,*9 , and *6 alleles and the P35372 A118G variant was determined by SNP genotyping . P20813 *4 , *9 , and *6 alleles were found to be associated with higher postmortem methadone concentrations in blood ( P < or = 0.05 ) . P35372 A118G was also associated with higher postmortem methadone concentrations in blood but not to a level of statistical significance ( P = 0.39 ) . In these methadone-related deaths , P35372 118GA was associated with higher postmortem benzodiazepine concentrations ( P = 0.04 ) , a finding not associated with morphine-related deaths . The risk of a methadone-related fatality during treatment may be evaluated in part by screening for P20813 *6 and A118G . Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo P62158 . P03950 is one of the most potent angiogenesis-inducing proteins . Angiostatin is one of the most potent angiogenesis inhibitors , and it contains the first four kringle domains of plasminogen ( P04264 -4 ) . Recombinant human plasminogen kringle 1-3 ( rK1-3 ) was expressed in Escherichia coli and purified to homogeneity . The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence . rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3 . The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane ( P62158 ) assay . Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis , and the equilibrium dissociation constants for the complex , Kd , are 0.89 and 0.18 microM , respectively . rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo P62158 in a concentration-dependent manner . These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin . Zinc-fingers and homeoboxes ( ZHX ) 2 , a novel member of the ZHX family , functions as a transcriptional repressor . Zinc-fingers and homeoboxes ( ZHX ) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y ( P23511 ) . Using a yeast two-hybrid system , a novel ubiquitous transcription factor Q9Y6X8 as a Q9UKY1 -interacting protein was cloned . Q9Y6X8 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains ( HDs ) as well as Q9UKY1 . The mRNA is expressed among various tissues . Q9Y6X8 not only forms a heterodimer with Q9UKY1 , but also forms a homodimer . Moreover , Q9Y6X8 interacts with the activation domain of P23511 . Further analysis revealed that Q9Y6X8 is a transcriptional repressor that is localized in the nuclei . Since Q9Y6X8 shares a number of properties in common with Q9UKY1 , we conclude that all these come under the ZHX family . The minimal functional domains of Q9Y6X8 were then characterized . The dimerization domain with both Q9UKY1 and Q9Y6X8 is the region containing HD1 , the domain that interacts with P23511 is the HD1 to Q92769 region , the repressor domain is the HD1 to a proline-rich region . Lastly , using an immunoprecipitation assay , we showed that Q9Y6X8 intrinsically interacts with P23511 in P29320 -293 cells and that Q9Y6X8 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells . Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network . Protective effects of metallothionein on isoniazid and rifampicin-induced hepatotoxicity in mice . Isoniazid ( DB00951 ) and DB01045 ( RFP ) are widely used in the world for the treatment of tuberculosis , but the hepatotoxicity is a major concern during clinical therapy . Previous studies showed that these drugs induced oxidative stress in liver , and several antioxidants abated this effect . Metallothionein ( MT ) , a member of cysteine-rich protein , has been proposed as a potent antioxidant . This study attempts to determine whether endogenous expression of MT protects against DB00951 and RFP-induced hepatic oxidative stress in mice . Wild type ( MT+/+ ) and MT-null ( MT-/- ) mice were treated intragastrically with DB00951 ( 150 mg/kg ) , RFP ( 300 mg/kg ) , or the combination ( 150 mg/kg DB00951 +300 mg/kg RFP ) for 21 days . The results showed that MT-/- mice were more sensitive than MT+/+ mice to DB00951 and RFP-induced hepatic injuries as evidenced by hepatic histopathological alterations , increased serum Q9NRA2 levels and liver index , and hepatic oxidative stress as evidenced by the increase of MDA production and the change of liver antioxidant status . Furthermore , DB00951 increased the protein expression of hepatic P05181 and DB00951 /RFP ( alone or in combination ) decreased the expression of hepatic P05177 . These findings clearly demonstrate that basal MT provides protection against DB00951 and RFP-induced toxicity in hepatocytes . The P05181 and P05177 were involved in the pathogenesis of DB00951 and RFP-induced hepatotoxicity . Expression of two variants of the human mu opioid receptor mRNA in SK-N-SH cells and human brain . A partial mu opioid receptor gene was isolated from a human genomic library using a mouse delta opioid receptor cDNA as a probe . Using information from this genomic clone and the published human mu receptor , P35372 , a cDNA was isolated from SK-N-SH mRNA that codes for a variant of the P35372 mRNA , MOR1A . The presence of MOR1A is also shown in human brain using RT-PCR . MOR1A differs from P35372 in that the 3' terminal intron has not been removed . An in-frame termination codon is found four amino acids after the 5' consensus splice site , making MOR1A eight amino acids shorter than P35372 . Both receptors show similar ligand binding and coupling to DB02527 in CHO- P04264 cells . The C-terminal differences between P35372 and MOR1A could have effects on receptor coupling or receptor transport and localization . Drug-induced activation of SREBP-controlled lipogenic gene expression in CNS-related cell lines : marked differences between various antipsychotic drugs . BACKGROUND : The etiology of schizophrenia is unknown , but neurodevelopmental disturbances , myelin- and oligodendrocyte abnormalities and synaptic dysfunction have been suggested as pathophysiological factors in this severe psychiatric disorder . DB04540 is an essential component of myelin and has proved important for synapse formation . Recently , we demonstrated that the antipsychotic drugs clozapine and haloperidol stimulate lipogenic gene expression in cultured glioma cells through activation of the sterol regulatory element-binding protein ( SREBP ) transcription factors . We here compare the action of chlorpromazine , haloperidol , clozapine , olanzapine , risperidone and ziprasidone on SREBP activation and SREBP-controlled gene expression ( ACAT2 , P04035 , Q01581 , P14324 , O75845 , Q9UBM7 , P01130 , P49327 and SCD1 ) in four CNS-relevant human cell lines . RESULTS : There were marked differences in the ability of the antipsychotic drugs to activate the expression of SREBP target genes , with clozapine and chlorpromazine as the most potent stimulators in a context of therapeutically relevant concentrations . Glial-like cells ( GaMg glioma and CCF-STTG1 astrocytoma cell lines ) displayed more pronounced drug-induced SREBP activation compared to the response in Q9UL51 human cortical neurons and SH-SY5Y neuroblastoma cells , indicating that antipsychotic-induced activation of lipogenesis is most prominent in glial cells . CONCLUSION : Our present data show a marked variation in the ability of different antipsychotics to induce SREBP-controlled transcriptional activation of lipogenesis in cultured human CNS-relevant cells . We propose that this effect could be relevant for the therapeutic efficacy of some antipsychotic drugs . [ Cell cycle analysis of endometrial cancer cells in vitro treated with growth factor and steroid hormone ] . The aim of this study was to overtake the mechanism of the control system in endometrial cancer cell line in vitro . Ishikawa cell ( IK cell ) and O14777 -1 cell ( O14777 cell ) derived from endometrial cancers were cultured with serum free medium ( SFM-101 ) . IK cell possessed P03372 ( ER ) , P06401 ( PR ) , Epidermal growth factor ( P01133 ) and its receptor ( P00533 ) . O14777 cell had PR , P01133 , and P00533 , however O14777 cell did not keep ER . P01133 stimulated the growth of IK cell , but the growth of O14777 cell was not stimulated by P01133 . S phase cells were increased by P01133 in IK cell , but were not increased by P01133 in O14777 cell . The growth of IK cell was stimulated significantly by P01133 and Estradiol-17 beta ( E2 ) + P01133 than control . However , E2+ P01133 did not stimulate the growth of IK cell than P01133 significantly . DB01406 ( D ) and D+ P01133 inhibited the growth of IK cell significantly than control . S phase cells were decreased by the treatment of D and D+ P01133 . From our results , P01133 stimulated the growth of ER positive endometrial cancer cell , but P01133 did not stimulate ER negative endometrial cancer cell . E2+ P01133 and P01133 stimulated the growth of IK cell as a same . However , D inhibited the growth of IK cell that was stimulated by P01133 . DB00688 impairs transduction of single-stranded adeno-associated viral vectors . Adeno-associated virus ( AAV ) liver-directed gene therapy seems a feasible treatment for Crigler-Najjar syndrome type I , an inherited liver disorder characterized by severe unconjugated hyperbilirubinemia . Transient immunosuppression coupled with vector administration seems needed to overcome host immune responses that prevent long-term expression in patients . The immunosuppressive mycophenolate mofetil ( DB00688 ) , which inhibits de novo synthesis of purines , is a promising candidate . To investigate the potential use of DB00688 in patients with Crigler-Najjar syndrome , we studied its effect on single-stranded AAV ( ssAAV ) -mediated correction of hyperbilirubinemia in the relevant preclinical model , the Gunn rat . Although DB00688 was well tolerated and effective it also impaired the efficacy of ssAAV . Subsequent in vitro studies showed that this effect is not specific for P22309 deficiency . In fact , clinical relevant concentrations of mycophenolic acid ( DB00603 ) , the active compound of DB00688 , also impair the transduction of P29320 -293T cells by ssAAV . Because this effect was reversed by guanosine addition , it seems that intracellular levels of this nucleotide become limited , suggesting that DB00603 impairs second-strand DNA synthesis . This is corroborated by observations that DB00603 did not impair transduction of 293T cells by a self-complementary AAV ( scAAV ) vector and that DB00688 did not reduce the scAAV efficacy in the Gunn rat . In conclusion , DB00688 impairs ssAAV-mediated liver-directed gene therapy , which is relevant for the use of this immunosuppressive agent with single-stranded vectors . Furthermore , because this effect is due to impaired second-strand synthesis , the use of DB00688 with scAAV seems warranted . 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 . The agonist activity of tamoxifen is inhibited by the short heterodimer partner orphan nuclear receptor in human endometrial cancer cells . Short heterodimer partner ( Q15466 ) is an orphan nuclear receptor that interacts with ER(alpha) and ERbeta and inhibits E2-induced transcription . We examined how Q15466 affects tamoxifen 's estrogen agonist activity in endometrial cells . We report that Q15466 interacts with 4-hydroxytamoxifen ( DB04468 ) or E2-occupied ER(alpha) in a temperature-dependent manner in vitro . In transient transfection assays , Q15466 inhibited DB04468 -stimulated reporter gene activity from an estrogen response element ( ERE ) in ER-positive RL95-2 but not in O14777 -1A human endometrial carcinoma cells transfected with ER(alpha) or ERbeta . Q15466 inhibited E2-induced transcriptional activity in ER(alpha)- or ERbeta-transfected O14777 -1A or Chinese hamster ovary- P04264 cells . Q15466 inhibition of E2 activity was greater for ER(alpha) than ERbeta from the nonpalindromic ERE in the pS2 gene promoter in Chinese hamster ovary- P04264 but not O14777 -1A cells . Thus , ER subtype , cell type , and ERE sequence influence Q15466 repressor activity . An ER(alpha) mutant lacking activator function-1 showed reduced inhibition by Q15466 . In glutathione S-transferase pull-down experiments , Q15466 inhibited ER(alpha) dimerization , providing a possible mechanism to account for the inhibitory effect of Q15466 on ER activity . These results identify Q15466 as novel target for blocking DB04468 agonist activity in endometrial cells . Systems pharmacology assessment of the 5-fluorouracil pathway . AIM : To assess the impact of the 5-fluorouracil ( DB00544 ) drug-pathway genes on cytotoxicity , and determine whether loss-of-function analyses coupled with functional assays can help prioritize pharmacogenomic candidate genes . MATERIALS & METHODS : Dose-response experiments were used to quantify the phenotype of sensitivity to DB00544 following the specific knockdown of genes selected from the DB00544 PharmGKB drug pathway in three human colorectal cell lines . Changes in sensitivity were considered significant if the IC(50) for shRNA-exposed cells were three standard deviations outside the mean IC(50) for control-treated cells . RESULTS : Of the 24 genes analyzed , 13 produced significant changes on the phenotype of sensitivity to DB00544 ( P00374 , Q14117 , P23919 , P33316 , Q05932 , Q92820 , P15531 , Q8TCD5 , P23921 , P04818 , Q9BZX2 , P13051 and P11172 ) . CONCLUSION : The RNAi screening strategy enabled prioritization of the genes from the DB00544 drug pathway . Further validation of the genes credentialed in this study should include gene activity or expression and mutation analyses of clinical samples . 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 . DB01645 potentiates the P01160 effect on a K(+)-conductance in P29320 -293 cells . P29320 -293 cells are known to reflect many features of the late distal tubule . Furthermore , they have the ability to release urodilatin , the structural analog to P01160 . RT-PCR was performed to test for the expression of natriuretic peptide receptors . While the mRNA for the human P01160 receptor ( P16066 , P16066 ) could be amplified , the P09543 -specific receptor P20594 ( P20594 ) and the receptor specific for guanylins , P25092 , could not be detected . In patch clamp experiments the effects of P01160 ( 10 nM ) on membrane voltage ( V(m) ) were monitored and P29320 -293 cells depolarized by 2.3 +/- 0.5 mV ( n=14 ) . In the presence of the P01133 receptor blocker genistein ( 10 microM ) the effect of P01160 was increased by 65 % to 3.9 +/- 0.8 mV ( n=14 ) . After removal of genistein the P01160 -mediated depolarization further increased by 147 % to 5.7 +/- 1.0 mV ( n=14 ) . P01160 given repetitively without genistein had no increasing depolarizing effect in P29320 -293 cells with time . The P01160 effect could be fully blocked by 1 mM Ba(2+) and by 1 microM of the specific PKG inhibitor KT5823 indicating that P01160 inhibits a K(+)-conductance via a cGMP-dependent protein kinase . DB01645 itself hyperpolarized the membrane voltage of P29320 -293 cells by -3.9 +/- 0.6 mV ( n=11 ) and this effect could also be fully blocked by Ba(2+) ( -0.3 +/- 0.1 mV , n=5 ) , indicating that genistein activates a K(+)-conductance which contributes significantly to the membrane potential of P29320 -293 cells . Atrial natriuretic peptide binds to P01160 - Q96GN5 receptors in neuroblastoma cells or is degraded extracellularly at the DB00133 - DB00120 bond . P01160 - Q96GN5 receptors for atrial natriuretic peptide ( P01160 ) showed the following rank order of affinity in intact human neuroblastoma cells NB-OK-1 : human P01160 -(99-126) approximately human P01160 -(102-126) approximately rat P01160 -(99-126) ( P04264 17-32 pM ) > human P01160 -(103-126) > porcine brain natriuretic peptide ( DB04899 ) . Analogues truncated at the C-terminal extremity or devoid of a disulphide bridge , such as rat P01160 -(103-123) , rat C- P01160 -(102-121) , rat P01160 -(111-126) , rat P01160 -(99-109) and rat [desCys105,Cys121] P01160 -(104-126) and chicken P23582 , were not recognized . The occupancy of these high affinity P01160 - Q96GN5 receptors led to marked cyclic GMP accumulation in the presence of 3-isobutyl 1-methylxanthine . An ectoenzymic activity , partly shed in the incubation medium , provoked the stepwise release of DB00120 - DB00125 -[125I] DB00135 , DB00125 -[125I] DB00135 and [125I] DB00135 from rat [125I] P01160 -(99-126) , at an optimal pH of 7.0 . Its inhibition by 1,10-phenanthroline , DB00974 and bacitracin but not by thiorphan suggests the contribution of at least one neutral metalloendopeptidase , distinct from EC 3.4.24.11 , for which P01160 showed high affinity . 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 . Small interfering RNA knocks down the molecular target of alendronate , farnesyl pyrophosphate synthase , in osteoclast and osteoblast cultures . P14324 ( FPPS ) , an enzyme in the mevalonate pathway , is the inhibition target of alendronate , a potent FDA-approved nitrogen-containing bisphosphonate ( N-BP ) drug , at the molecular level . DB00630 not only inhibits osteoclasts but also has been reported to positively affect osteoblasts . This study assesses the knockdown effects of siRNA targeting FPPS compared with alendronate in both osteoclast and osteoblast cultures . Primary murine bone marrow cell-induced osteoclasts and the preosteoblast MC3T3-E1 cell line were used to assess effects of anti-FPPS siRNA compared with alendronate . Results show that both FPPS mRNA message and protein knockdown in serum-based culture is correlated with reduced osteoclast viability . FPPS siRNA is more potent than 10 μM alendronate , but less potent than 50 μM alendronate on reducing osteoclast viability . Despite FPPS knockdown , no significant changes were observed in osteoblast proliferation . FPPS knockdown promotes osteoblast differentiation significantly but not cell mineral deposition . However , compared with 50 μM alendronate dosing , FPPS siRNA does not exhibit cytotoxic effects on osteoblasts while producing significant effects on ostoblast differentiation . Both siRNA and alendronate at tested concentrations do not have significant effects on cultured osteoblast mineralization . Overall , results indicate that siRNA against FPPS could be useful for selectively inhibiting osteoclast-mediated bone resorption and improving bone mass maintenance by influencing both osteoclasts and osteoblasts in distinct ways . Characterization of the inhibitory effects of erythromycin and clarithromycin on the Q12809 potassium channel . Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans , however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking . The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current ( IKr ) encoded by Q12809 ( the human ether-a-go-go-related gene ) at drug concentrations , temperature and ionic conditions mimicking those occurring in human subjects . DB01345 currents in P29320 293 cells stably transfected with Q12809 were recorded using a whole cell voltage clamp method . Exposure of cells to erythromycin reduced the Q12809 encoded potassium current in a concentration dependent manner with an IC50 of 38.9 +/- 1.2 microM and Hill Slope factor of 0.4 +/- 0.1 . DB01211 produced a similar concentration-dependent block with an IC50 of 45.7 +/- 1.1 microM and Hill Slope factor of 1.0 +/- 0.1 . Erythromycin ( 25-250 microM ) and clarithromycin ( 5 or 25 microM ) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol . The results of this study document that both erythromycin and clarithromycin significantly inhibit the Q12809 potassium current at clinically relevant concentrations . Lectin-like oxidized P01130 -1 ( P78380 ) supports adhesion of mononuclear leukocytes and a monocyte-like cell line THP-1 cells under static and flow conditions . Adhesion of mononuclear leukocytes to vascular endothelial cells appears one of the initial steps in the process of atherogenesis and inflammation . We examined if P78380 , an endothelial scavenger receptor with C-type lectin-like structure , can support adhesion of mononuclear leukocytes . Under a static condition , CHO- P04264 cells stably expressing P78380 showed more prominent adhesion of human peripheral blood mononuclear leukocytes and THP-1 cells than untransfected CHO- P04264 cells , in a temperature-independent fashion . Mononuclear leukocytes also adhered to plastic plates precoated with recombinant soluble P78380 extracellular domain . A neutralizing anti- P78380 monoclonal antibody , as well as oxidized low-density lipoprotein , significantly blocked adhesion of THP-1 cells to CHO- P04264 cells overexpressing P78380 and bovine aortic endothelial cells . Under a flow condition , increased numbers of THP-1 cells showed rolling with reduced velocities on P78380 -expressing CHO- P04264 cells , compared with those on untransfected CHO- P04264 cells . Taken together , P78380 can work as a cell surface receptor for mononuclear leukocytes under both static and flow conditions . 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 . A novel tumor necrosis factor-responsive transcription factor which recognizes a regulatory element in hemopoietic growth factor genes . A conserved DNA sequence element , termed cytokine 1 ( P04264 ) , is found in the promoter regions of many hemopoietic growth factor ( P14210 ) genes . Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor , NF-GMa , which is a protein with a molecular mass of 43 kilodaltons . It interacts with different affinities with the P04264 -like sequence from a number of P14210 genes , including granulocyte macrophage colony-stimulating factor ( GM- P04141 ) , granulocyte ( G ) - P04141 , interleukin 3 ( P08700 ) , and P05113 . We show here that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor-alpha ( P01375 ) treatment and that the P04264 sequence from the G- P04141 gene is a P01375 -responsive enhancer in these cells . The NF-GMa protein is distinct from another P01375 -responsive transcription factor , NF-kappa B , by several criteria . Firstly , several NF-kappa B-binding sites , although having sequence similarity with the P04264 sequence , can not compete efficiently for NF-GMa binding to P04264 . Secondly , the P04264 sequence from both G- P04141 and GM- P04141 does not respond to phorbol ester treatment as would an NF-kappa B-binding element . These results demonstrate that NF-GMa is a novel transcription factor inducible by P01375 and binds to a common element in P14210 gene promoters . [ Measurement of rifampicin and clarithromycin in serum by high-performance liquid chromatography with electrochemical detection ] . DB01045 ( RFP ) induces hepatic drug-metabolizing enzymes , making drug interactions a very important clinical problem . DB01211 ( P62158 ) metabolism is reportedly enhanced by induction of hepatic drug-metabolizing enzymes ( P08684 ) by RFP , so that the blood lend of P62158 decreases when RFP is administered concurrently . We connected an electrochemical detector to a high-performance liquid chromatograph ( HPLC ) for simple , rapid , easy measurement of blood concentrations of RFP and P62158 . Using samples of patient serum , normal serum , and reference standards , we compared HPLC by an external laboratory and the results of LC/MS/MS analysis with those of this new assay . A strong correlation was seen between our HPLC results and those of the external laboratory in RFP levels ( r=0.975 , p < 0.01 ) . A strong correlation was also seen between results we obtained for P62158 with the electrochemical detector in this assay and values measured by LC/MS/MS analysis ( r=0.995 , p < 0.01 ) . Our method enabled simple , rapid measurement of RFP and P62158 by connecting the HPLC and electrochemical detector in tandem . This system was used to modulate dosage during combined therapy with RFP and P62158 . The therapeutic effect for nontuberculous mycobacteriosis is expected to improve , and our HPLC is expected to be useful for simple , rapid , easy measurement of blood concentrations . Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes . DB03823 gallate ( EGCG ) inhibits drug metabolizing enzymes by unknown mechanisms . Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates . EGCG was incubated with rat liver microsomes at 1-100μM for 30min . The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase ( P04406 ) , actin , cytochrome P450 ( CYP ) 1A1 , P05177 , CYP2B1/2 , P05181 , CYP3A , catechol-O-methyltransferase ( P21964 ) and microsomal glutathione transferase 1 ( P10620 ) . All but actin and soluble P21964 were positively detected at ≥1μM EGCG , indicating EGCG selectively bound to a subset of proteins including membrane-bound P21964 . The binding correlated well with inhibition of CYP activities , except for P05181 whose activity was unaffected despite evident binding . The antioxidant enzyme P10620 , but not cytosolic GSTs , was remarkably inhibited , providing novel evidence supporting the pro-oxidative effects of EGCG . When microsomes incubated with EGCG were probed on Western blots , all but the actin and P05181 antibodies showed a significant reduction in binding at ≥1μM EGCG , suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins . This raised the possibility that previous reports on EGCG regulating protein expression using P04406 as a reference should be revisited for accuracy . Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels . EGCG effects were partially abolished in the presence of 1mM glutathione , suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults . 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 . [ Innate resistance to thymidylate synthase inhibition after 5-fluorouracil treatment -- a rationale of combined use of cisplatin and its optimal administration dose ] . We examined the changes of the number of DB00322 MP binding sites of thymidylate thynthase ( TS-BS ) in Yoshida sarcoma after administration of DB00544 to the tumor bearing rats . We also investigated the optimal dose of DB00515 for the increase of intracellular folate level . In the group received consecutive 7-days administration of DB09327 ( U-7 group ) , total TS-BS was significantly increased compared with non-treatment group and the group received only DB09327 ( U-1 group ) . For free TS-BS , however , there was no difference despite of DB09327 administration . P04818 inhibition rate ( TSIR ) was , therefore , significantly high in U-7 group compared with U-1 group . It seemed necessary to take some counter measure for the induction of TS in the tumor tissue when DB00544 chemotherapy was performed . The optimal dose of DB00515 as a modulator of DB00544 was 1 mg/kg in rat when it was estimated from the changes of intracellular folate levels after administration , which was less than the dose to reveal its own anticancer effect . Induced segregation of human syntenic genes by 5-bromodeozyuridine + near-visible light . Chromosome-breaking agents have been used in two different procedures for promoting segregation of syntenic genes on human chromosome 12 . In method A , a human-Chinese hamster cell hybrid containing the single human chromosome 12 was treated either with 5-bromodeoxyuridine BrdU + near-visible light or with X-rays . In method B , normal human fibroblasts were treated with BrdU + near-visible light followed by their fusion with a Chinese hamster glycine-requiring cell mutant CHO- P04264 /gly-A . Since the human complementing gene for serine hydroxymethyltransferase , an enzyme deficient in gly-A , lies on human chromosome 12 , only those hybrids retaining that chromosome can survive the glycine-free medium . Clones isolated from both procedures were analyzed for the loss or retention of four other syntenic genes on chromosome 12 , P60174 , P04406 , LDH B , and PepB . The results demonstrate that method B is much more effective in generating clones with extensive marker losses . In addition , the segregation pattern and frequency obtained in this study provided information on the linear order of P60174 and P04406 on chromosome 12 . Effects of systemic injections of vilazodone , a selective serotonin reuptake inhibitor and serotonin 1A receptor agonist , on anxiety induced by predator stress in rats . We examined the effect of DB06684 , a selective serotonin reuptake inhibitor ( SSRI ) and serotonin 1A ( 5-HT(1A) ) receptor agonist [ Bartoszyk , G.D. , Hegenbart , R. , Ziegler , H. , 1997. P50402 68843 , a serotonin reuptake inhibitor with selective presynaptic P08908 receptor agonistic properties. Eur. J. Pharmacol. 322 , 147-153. ] , on change in affect following predator stress . DB06684 and vehicle injection ( intraperitoneal ) occurred either 10 min after predator stress ( prophylactic testing ) , or 90 min prior to behavioral testing for the effects of predator stress ( therapeutic testing ) . Predator stress involved unprotected exposure of rats to a domestic cat . Behavioral effects of stress were evaluated with hole board , plus-maze , and acoustic startle tests 1 week after stress . Predator stress increased anxiety-like behavior in the plus-maze and elevated response to acoustic startle . In prophylactic testing , DB06684 affected stress potentiation of startle at doses above 5 mg/kg . DB06684 increased stress elevation of startle at 10 mg/kg . Higher doses of DB06684 ( 20 and 40 mg/kg ) blocked stress potentiation of startle . In contrast , DB06684 had no effect on stress potentiation of anxiety in the plus-maze . In therapeutic testing , DB06684 increased stress elevation of startle at all doses . In contrast , therapeutic DB06684 had no effect on stress potentiation of anxiety in the plus-maze . Taken together , the data suggest a prophylactic potential for DB06684 in the treatment of changes in hypervigilance following severe stress . Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors . We have recently shown that the mu-opioid receptor [ P35372 , also termed mu-opioid peptide ( MOP ) receptor ] is associated with the phospholipase D2 ( O14939 ) , a phospholipid-specific phosphodiesterase located in the plasma membrane . We further demonstrated that , in human embryonic kidney ( P29320 ) 293 cells co-expressing P35372 and O14939 , treatment with ( D-Ala2 , Me Phe4 , Glyol5 ) enkephalin ( DAMGO ) led to an increase in O14939 activity and an induction of receptor endocytosis , whereas morphine , which does not induce opioid receptor endocytosis , failed to activate O14939 . In contrast , a C-terminal splice variant of the mu-opioid receptor ( MOR1D , also termed MOP(1D) ) exhibited robust endocytosis in response to both DAMGO and morphine treatment . We report here that MOR1D also mediates an agonist-independent ( constitutive ) O14939 -activation facilitating agonist-induced and constitutive receptor endocytosis . Inhibition of O14939 activity by over-expression of a dominant negative O14939 ( nPLD2 ) blocked the constitutive O14939 activation and impaired the endocytosis of MOR1D receptors . Moreover , we provide evidence that the endocytotic trafficking of the delta-opioid receptor [ Q8IXH6 , also termed delta-opioid peptide ( DOP ) receptor ] and cannabinoid receptor isoform 1 ( P21554 ) is also mediated by a O14939 -dependent pathway . These data indicate the generally important role for O14939 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor ( GPCR ) endocytosis . Estrogenic chemicals at puberty change ERalpha in the hypothalamus of male and female rats . The effects of two environmental endocrine disruptors , the synthetic pharmaceutical estrogen DB00977 ( EE ) and bisphenol-A ( Q03001 ) , were analysed in male and female rats in a very sensitive developmental period , puberty . Immunohistochemistry was used to evaluate changes in the number of cells expressing estrogen receptors ( P03372 ) in the arcuate nucleus ( ARC ) , ventromedial nucleus ( VMH ) and medial preoptic area ( DB00603 ) of the hypothalamus . Animals were treated during early puberty , from P01160 23 to P01160 30 , with EE and Q03001 given orally every day . They were then sacrificed and perfused on P01160 37 or P01160 90 , and blood and brains were collected for hormonal determination ( testosterone and estradiol ) and immunohistochemistry ( estrogen receptors , ER ) . At P01160 37 , ER-labelled neurons were higher in males than in females in the ARC and DB00603 . EE and Q03001 increased ER-labelled neurons in the ARC and DB00603 . At P01160 90 , females showed higher ER-labelled neurons in the VMH . EE and Q03001 increased ER-labelled neurons in the DB00603 in females . EE increased testosterone in males at P01160 37 and estradiol in females at P01160 90 . These results indicate the ability of estrogenic chemicals to change the reproductive neural circuits during puberty in male and female rats . Self-assembly of P29320 cell-secreted ApoE particles resembles ApoE enrichment of lipoproteins as a ligand for the P01130 -related protein . Recent studies have shown that the lipidation and assembly state of apolipoprotein E ( apoE ) determine receptor recognition and amyloid-beta peptide ( Abeta ) binding . We previously demonstrated that apoE secreted by P29320 cells stably expressing apoE3 or apoE4 ( P29320 -apoE ) binds Abeta and inhibits Abeta-induced neurotoxicity by an isoform-specific process that requires apoE receptors . Here we characterized the structure of P29320 -apoE assemblies and determined their receptor binding specificity . By chromatography , P29320 -apoE elutes in high molecular mass fractions and is the size of plasma HDL , consistent with a multiprotein assembly . No lipid was associated with these apoE assemblies . Several methods for analyzing receptor binding indicate that P29320 -apoE is a ligand for low-density lipoprotein ( LDL ) receptor-related protein ( Q14764 ) but not the P01130 . This suggests that self-assembly of apoE may induce a functional conformation necessary for binding to Q14764 . Our results indicate that , in addition to lipid content , the assembly state of apoE influences Abeta binding and receptor recognition . The infection of human primary cells and cell lines by human cytomegalovirus : new tropism and new reservoirs for HCMV . Human cytomegalovirus ( HCMV ) infection is asymptomatic in common persons and could reactive in immunosuppression groups . HCMV was considered as endothelial cells ( EC ) tropism and leukocyte tropism . We hypothesized that HCMV will infect other cell types from human which have not been reported yet . The HCMV released from human MRC-5 was inoculated into eight human primary cells and cell lines , including human dermal fibroblasts ( HDF ) , human embryo-chondrocytes ( O14777 ) , human embryo-myoblasts ( HEM ) , and human embryo-kidney endothelial cell ( P29320 -EC ) , human marrow stromal cell ( HMSC ) . The cell lines were ECV304 , Chung liver cell and L02 . Several detection methods specific for HCMV , in which PCR for HCMV DNA sequences , immunofluorescence for pp65 antigen , Western-blot for gB protein , as well as cytopathic effect observation were conducted at different time post-infection . The results indicated that four cells in our experiment ( HDF , HEM , O14777 and HMSC ) were HCMV-positive . The occurring time of cytopathic effect was different in these four cells . Our experiment found the new tropism and new reservoirs for HCMV .