interacts_with DB00563?
P50750 regulates AR promoter selectivity and cell growth through serine 81 phosphorylation . Previously we determined that S81 is the highest stoichiometric phosphorylation on the androgen receptor ( AR ) in response to hormone . To explore the role of this phosphorylation on growth , we stably expressed wild-type and S81A mutant AR in LHS and LAPC4 cells . The cells with increased wild-type AR expression grow faster compared with parental cells and S81A mutant-expressing cells , indicating that loss of S81 phosphorylation limits cell growth . To explore how S81 regulates cell growth , we tested whether S81 phosphorylation regulates AR transcriptional activity . LHS cells stably expressing wild-type and S81A mutant AR showed differences in the regulation of endogenous AR target genes , suggesting that S81 phosphorylation regulates promoter selectivity . We next sought to identify the S81 kinase using ion trap mass spectrometry to analyze AR-associated proteins in immunoprecipitates from cells . We observed cyclin-dependent kinase (CDK)9 association with the AR . P50750 phosphorylates the AR on S81 in vitro . Phosphorylation is specific to S81 because P50750 did not phosphorylate the AR on other serine phosphorylation sites . Overexpression of P50750 with its cognate cyclin , P12004 T , increased S81 phosphorylation levels in cells . Small interfering RNA knockdown of P50750 protein levels decreased hormone-induced S81 phosphorylation . Additionally , treatment of LNCaP cells with the P50750 inhibitors , 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole and DB03496 , reduced S81 phosphorylation further , suggesting that P50750 regulates S81 phosphorylation . Pharmacological inhibition of P50750 also resulted in decreased AR transcription in LNCaP cells . Collectively these results suggest that P50750 phosphorylation of AR S81 is an important step in regulating AR transcriptional activity and prostate cancer cell growth . ICE/ P29466 inhibitors as novel anti-inflammatory drugs . In recent years , several strategies that selectively inhibit pro-inflammatory cytokines , have yielded effective protein-based therapies for inflammatory disorders , validating the therapeutic hypothesis that intervention in cytokine signalling can provide clinical benefit . However , these protein-based products must be administered by injection , a constraint associated with inconvenience , adverse effects and expense for patients , caregivers and insurers . Besides interfering with the effects of cytokines such as P01375 or IL-1beta that have already been produced , inhibition of pro-inflammatory cytokine production or signalling with low-molecular weight orally-active drugs would combine the convenience of conventional pharmaceuticals with the focused efficacy of the protein therapies . Reducing IL-1beta and Q14116 production by inhibition of IL-1beta converting enzyme ( ICE , caspase-1 ) is one promising strategy because of the key roles of these cytokines in many inflammatory diseases . DB04875 , the first orally available , potent and selective ICE inhibitor to enter clinical trials , is currently under investigation in rheumatoid arthritis . 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 . Endothelial dihydrofolate reductase : critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase . Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin ( H4B ) may cause uncoupling of endothelial nitric oxide synthase ( P29474 ) to produce superoxide ( O2*- ) . H4B was found recyclable from its oxidized form by dihydrofolate reductase ( P00374 ) in several cell types . Functionality of the endothelial P00374 , however , remains completely unknown . Here we present findings that specific inhibition of endothelial P00374 by RNA interference markedly reduced endothelial H4B and nitric oxide ( NO. ) bioavailability . Furthermore , angiotensin II ( 100 nmol/liter for 24 h ) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of P00374 . This response was associated with a significant increase in endothelial O2*- production , which was abolished by P29474 inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase , strongly suggesting H2O2-dependent P29474 uncoupling . Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O2* ( Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated P03372 O2*- signal at 30 min ) in response to angiotensin II , preceding a second peak in O2*- production at 24 h that predominantly depended on uncoupled P29474 . Overexpression of P00374 restored NO. production and diminished P29474 production of O2*- in angiotensin II-stimulated cells . In conclusion , these data represent evidence that P00374 is critical for H4B and NO. bioavailability in the endothelium . Endothelial NAD(P)H oxidase-derived H2O2 down-regulates P00374 expression in response to angiotensin II , resulting in H4B deficiency and uncoupling of P29474 . This signaling cascade may represent a universal mechanism underlying P29474 dysfunction under pathophysiological conditions associated with oxidant stress . Differences in hepatotoxicity and gene expression profiles by anti-diabetic Q07869 gamma agonists on rat primary hepatocytes and human HepG2 cells . Agonists of peroxisome proliferator-activated receptor gamma ( PPARgamma ) are a new class of oral drugs designed to treat insulin-resistant diabetes ( i.e. , type 2 diabetes ) . However , troglitazone , the first compound in the class approved by the US Food and Drug Administration ( FDA ) in 1997 was found to be hepatotoxic and was withdrawn from the market after reports of severe liver failure . The mechanism of Q07869 gamma agonist-induced hepatotoxicity remains unknown . In this study , we examined the hepatotoxic effects of five Q07869 gamma agonists ( ciglitazone , pioglitazone , rosiglitazone , troglitazone , and DB04971 ) on rat primary hepatocytes and human HepG2 cells . We also compared the gene expression profiles of rat primary hepatocytes after exposure to Q07869 gamma agonists by using the Rat Genome Survey Microarray system from Applied Biosystems in order to understand the mechanisms of hepatotoxicities induced by PPARgamma agonists . Consistent with the hepatotoxicity data , our results demonstrate that the gene expression profiles affected by troglitazone and ciglitazone can be clearly distinguished from those by pioglitazone and rosiglitazone . Genes that are differentially expressed between the more toxic troglitazone/ciglitazone group and the less toxic rosiglitazone/pioglitazone group are involved in necrotic , apoptotic , and cell proliferative pathways . The five compounds were also clustered based on a set of molecular descriptors . The clustering based on chemical structural information is in good agreement with the clustering of compounds based on cytotoxicity or gene expression data , indicating a strong relationship between chemical structure and biological endpoints . Our work suggests that microarray analysis together with toxicological observations can be used to rank drugs for hepatotoxicity and to evaluate the safety of new compounds . Establishment of pemetrexed-resistant non-small cell lung cancer cell lines . DB00642 ( P15941 ) , a multitargeted antifolate with manageable toxicity , is active against non-squamous non-small cell lung cancer ; however , most patients eventually acquire resistance to P15941 . To elucidate the resistant mechanism , we established P15941 -resistant lung adenocarcinoma cell lines . Two parental cell lines , PC-9 and A549 , were treated with step-wise increasing concentrations of P15941 . Growth inhibition was determined by the 3-[4,5-dimethyl-thizol-2-yl]-2,5-diphenyltetrazolium bromide assay . Expression of the genes encoding thymidylate synthase ( TS ) , dihydrofolate reductase ( P00374 ) , and glycinamide ribonucleotide formyltransferase ( GARFT ) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction . The four PC-9 sublines were more resistant than the PC-9 cell line to P15941 ( 2.2- , 2.9- , 8.4- , and 14.3-fold , respectively ) . The four A549 sublines also showed more resistance to P15941 ( 7.8- , 9.6- , 42.3- , and 42.4-fold , respectively ) than the parent cell line . All resistant sublines showed cross-resistance to cisplatin , but not to docetaxel , vinorelbine , 5-fluorouracil , or the active metabolite of irinotecan , SN-38 . All P15941 -resistant sublines expressed more TS than the parental cells , by polymerase chain reaction and Western blotting . P00374 was significantly increased in the four P15941 -resistant A549 sublines . GARFT did not correlate with resistance to P15941 . In summary , P15941 -resistant cells remained sensitive to docetaxel , vinorelbine , 5-fluorouracil , and irinotecan . TS expression appeared to be associated with resistance to P15941 . Integrative analysis of transcriptomics , proteomics , and metabolomics data of white adipose and liver tissue of high-fat diet and rosiglitazone-treated insulin-resistant mice identified pathway alterations and molecular hubs . The incidences of obesity and type 2 diabetes are rapidly increasing and have evolved into a global epidemic . In this study , we analyzed the molecular effects of high-fat diet ( HFD ) -induced insulin-resistance on mice in two metabolic target tissues , the white adipose tissue ( WAT ) and the liver . Additionally , we analyzed the effects of drug treatment using the specific PPARγ ligand rosiglitazone . We integrated transcriptome , proteome , and metabolome data sets for a combined holistic view of molecular mechanisms in type 2 diabetes . Using network and pathway analyses , we identified hub proteins such as P21912 and P53597 in WAT and deregulation of major metabolic pathways in the insulin-resistant state , including the TCA cycle , oxidative phosphorylation , and branched chain amino acid metabolism . Rosiglitazone treatment resulted mainly in modulation via Q07869 signaling and oxidative phosphorylation in WAT only . Interestingly , in HFD liver , we could observe a decrease of proteins involved in vitamin B metabolism such as Q6P996 and P00374 and the according metabolites . Furthermore , we could identify sphingosine ( Sph ) and sphingosine 1-phosphate ( SP1 ) as a drug-specific marker pair in the liver . In summary , our data indicate physiological plasticity gained by interconnected molecular pathways to counteract metabolic dysregulation due to high calorie intake and drug treatment . DB01708 prevents the aggregation of platelets obtained from postmenopausal women with type 2 diabetes mellitus through the activation of the PKC/ P29474 /NO pathway . The steroid hormone dehydroepiandrosterone ( DB01708 ) , suggested to be a cardioprotector , prevents platelet aggregation in healthy humans . This hormone is reduced in postmenopausal women by 60 % of its normal value . Platelets in patients with type 2 diabetes ( T2D ) are more sensitive to aggregation , which has been attributed to a reduced ability to produce nitric oxide ( NO ) . In light of these precedents and considering that DB01708 is able to increase the production of NO in cultured endothelial cells , we suggest that DB01708 prevents the aggregation of platelet from postmenopausal women with T2D through the activation of PKC/ P29474 /NO/cGMP pathway . To determine the effect of DB01708 in platelet aggregation , platelet-rich plasma ( PRP ) obtained from postmenopausal women with T2D was preincubated with DB01708 , and aggregation induced by ADP was determined in the presence or absence of DB04223 ( LNG-nitroarginine ) , Rottlerin , NOS , or PKC delta inhibitors , respectively . Platelet NO production was measured with the fluorescent probe DAF2DA and P29474 activation was determined by Western blot , using an anti-p- P29474 ( ser 1177 ) antibody . DB01708 1 ) prevented platelet aggregation by 40 % compared to control , 2 ) increased NO production by 63 % , 3 ) increased p- P29474 ( phosphorylated endothelial nitric oxide synthase ) levels , and 4 ) increased cGMP production . These effects were reduced in the presence of DB04223 or Rottlerin . DB01708 prevents platelet aggregation induced by ADP . This effect is mediated by the activation of the PKCδ/ P29474 /NO/cGMP pathway . Our results suggest that DB01708 could be considered to be a potential therapeutic tool in the prevention of atherothrombotic processes in postmenopausal women with T2D . Reduced folate carrier and dihydrofolate reductase expression in acute lymphocytic leukemia may predict outcome : a Children 's Cancer Group Study . PURPOSE : DB00563 is a major component of current treatment regimens for children with acute lymphocytic leukemia ( ALL ) . Potential mechanisms of methotrexate resistance include impaired drug uptake , decreased drug retention , and dihydrofolate reductase ( P00374 ) amplification . The purpose of this study was to assess whether reduced folate carrier ( P41440 ) and P00374 expression in untreated leukemic blasts correlated with outcome . METHODS : Quantitative real-time RT-PCR was used to measure P41440 and P00374 mRNA expression in leukemic blasts from 40 newly diagnosed patients with ALL obtained in a blinded fashion from Children 's Cancer Group studies . RESULTS : Low P41440 expression at diagnosis correlated significantly with an unfavorable event free survival . Surprisingly , low , not high , P00374 expression correlated significantly with an unfavorable event-free survival . Proliferative cell nuclear antigen ( P12004 ) expression demonstrated a weak inverse relationship between sample P12004 and P00374 or P41440 expression , suggesting that P00374 and P41440 expression may be markers for factors other than drug resistance . CONCLUSIONS : These results suggest that impaired transport may be an important mechanism of intrinsic methotrexate resistance in ALL , and P00374 expression also may be an important prognostic factor in ALL . Additional studies are necessary to clarify the mechanism for the correlation of low P00374 expression with poor outcome . Microarray analysis revealed different gene expression patterns in HepG2 cells treated with low and high concentrations of the extracts of Anacardium occidentale shoots . In this study , the effects of low and high concentrations of the Anacardium occidentale shoot extracts on gene expression in liver HepG2 cells were investigated . From MTT assays , the concentration of the shoot extracts that maintained 50 % cell viability ( IC(50) ) was 1.7 mg/ml . Cell viability was kept above 90 % at both 0.4 mg/ml and 0.6 mg/ml of the extracts . The three concentrations were subsequently used for the gene expression analysis using Affymetrix Human Genome 1.0 S.T arrays . The microarray data were validated using real-time qRT-PCR . A total of 246 , 696 and 4503 genes were significantly regulated ( P < 0.01 ) by at least 1.5-fold in response to 0.4 , 0.6 and 1.7 mg/ml of the extracts , respectively . Mutually regulated genes in response to the three concentrations included CDKN3 , LOC100289612 , P00374 , Q99986 , Q99741 , Q96GD4 and P78334 . Genes like Q07973 , P38398 , O14965 , P06493 , P24941 , P11802 and P06213 were significantly regulated at 0.6 mg/ml and 1.7 mg but not at 0.4 mg/ml . However , the expression of genes including O75473 , P17936 , P06400 , P14735 , P01130 , P55157 , P04114 , MTIX , P04179 and P08294 were exclusively regulated at the IC(50) concentration . In conclusion , low concentrations of the extracts were able to significantly regulate a sizable number of genes . The type of genes that were expressed was highly dependent on the concentration of the extracts used . Cross-talk between PKA-Cβ and p65 mediates synergistic induction of Q07343 by roflumilast and NTHi . Phosphodiesterase 4B ( Q07343 ) plays a key role in regulating inflammation . DB01656 , a phosphodiesterase (PDE)4-selective inhibitor , has recently been approved for treating severe chronic obstructive pulmonary disease ( P48444 ) patients with exacerbation . However , there is also clinical evidence suggesting the development of tachyphylaxis or tolerance on repeated dosing of roflumilast and the possible contribution of Q07343 up-regulation , which could be counterproductive for suppressing inflammation . Thus , understanding how Q07343 is up-regulated in the context of the complex pathogenesis and medications of P48444 may help improve the efficacy and possibly ameliorate the tolerance of roflumilast . Here we show that roflumilast synergizes with nontypeable Haemophilus influenzae ( NTHi ) , a major bacterial cause of P48444 exacerbation , to up-regulate PDE4B2 expression in human airway epithelial cells in vitro and in vivo . Up-regulated PDE4B2 contributes to the induction of certain important chemokines in both enzymatic activity-dependent and activity-independent manners . We also found that protein kinase A catalytic subunit β ( PKA-Cβ ) and nuclear factor-κB ( NF-κB ) p65 subunit were required for the synergistic induction of PDE4B2 . PKA-Cβ phosphorylates p65 in a DB02527 -dependent manner . Moreover , Ser276 of p65 is critical for mediating the PKA-Cβ-induced p65 phosphorylation and the synergistic induction of PDE4B2 . Collectively , our data unveil a previously unidentified mechanism underlying synergistic up-regulation of PDE4B2 via a cross-talk between PKA-Cβ and p65 and may help develop new therapeutic strategies to improve the efficacy of DB05876 inhibitor . Overexpression of folate binding protein alpha is one of the mechanism explaining the adaptation of HT29 cells to high concentration of methotrexate . The human colon adenocarcinoma cell line HT29 can be adapted to 10(-7)- 10(-4) M concentrations of methotrexate ( MTX ) . Cells adapted to 10(-4) M MTX have an enterocyte-like phenotype with P00374 gene amplification . Presently , we hypothetized that an increased expression of folate binding protein ( FBP ) may participate to the MTX resistance of 10(-4) MTX HT29 cells . The cDNA FBPalpha/beta-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10(-4) and in 10(-7) M MTX HT29 respectively , than in standard type HT29 cells . An increase of transcript level was observed when decreasing folic acid concentration . PI- P98160 cleaved 7.7 times more membrane FBP in 10(-4) M than in 10(-7) M MTX and wild type HT29 cells . In contrast to 10(-7) M MTX cells , growth of 10(-4) M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 microM . In conclusion , the adaptive mechanism of HT29 cells resistant to 10(-4) M MTX is the result of the synergistic overexpression of both P00374 and FBPalpha . Overexpression of FBPalpha may be related to the enterocyte-like phenotype of the cells . DB00563 binds in a non-productive orientation to human dihydrofolate reductase in solution , based on NMR spectroscopy . P00374 ( P00374 ) is an intracellular target enzyme for folate antagonist drugs , including methotrexate . In order to compare the binding of methotrexate to human P00374 in solution with that observed in the crystalline state , NMR spectroscopy has been used to determine the conformation of the drug bound to human P00374 in solution . In agreement with what has been observed in the crystalline state , NOE 's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation . In contrast to what has been reported for E. coli P00374 in solution , only one bound conformation of methotrexate is observed . [ Genetic and clinical and pathological characteristics of breast cancer in premenopausal and postmenopausal women ] . This study involved 525 breast cancer ( BC ) patients of P24752 -4N0-2M0 stages at the age of 35 years and older . Significant differences in clinical and pathological characteristics between premenopausal and postmenopausal BC patients were found . Mostly marked differences were shown for positive lymph node correlation with distant metastasis , multicentric growth and local recurrence depending on menopause status . The prevalence of various morphological structures in primary tumors was appeared to be associated with different forms of tumor progression in pre- and postmenopausal women . We have studied polymorphisms in 15 genes involved in major cancer related pathways ( apoptosis , interleukins , folate metabolism enzymes genes ) . We found that variant genotypes of P42898 and P00374 genes were associated with an increased BC risk among premenopausal women while polymorphism in Q14116 , p53 genes were associated with BC among postmenopausal women . These results demonstrate novel biological information , which points the different mechanisms contributed to breast cancer progression in premenopausal and postmenopausal women . Suppression of tumor growth and metastasis by a P17948 antagonizing peptide identified from a phage display library . Although the P15692 -Flk-1-pathway has been known as the major driving force of angiogenesis , new evidence has shown that P17948 /Flt-1 plays important roles during the neovascularization under pathological conditions including tumor , atherosclerosis and arthritis . In search of Flt-1 receptor antagonizing peptides , we screened a phage display 12-mer-peptide library with recombinant Flt-1 protein . Seven candidate peptides were identified that specifically bound to P15692 receptor Flt-1 , of which peptide F56 ( WHSDMEWWYLLG ) almost abolished P15692 binding to receptor Flt-1 in vitro . In vivo , F56 fused with P00374 ( P00374 -F56 ) inhibited angiogenesis in a P62158 assay . Moreover , P00374 -F56 significantly inhibited the growth of nodules of human gastric cancer cell line MGC-803 in BALB/c nude mice . Histological analyses showed that necrosis of the implanted tumor was markedly enhanced following treatment with P00374 -F56 . In the severe combined immunodeficiency disease ( SCID ) mouse model for studying metastasis of the human breast cancer cell line BICR-H1 , synthetic peptide F56 significantly inhibited tumor growth and lung metastases . Taken together , our results have demonstrated that peptide F56 , as a Flt-1 receptor antagonist , fulfilled the antiangiogenic and antimetastatic effects by specifically interfering with the interaction between P15692 and receptor Flt-1 . Thus , short peptide F56 may have clinical potential in tumor therapy . P55157 inhibitor decreases plasma cholesterol levels in P01130 -deficient WHHL rabbits by lowering the VLDL secretion . To examine whether a microsomal triglyceride transfer protein ( P55157 ) -inhibitor is effective in patients with homozygous familial hypercholesterolemia , we administered ( 2S ) -2-cyclopentyl-2-[4-[(2,4-dimethyl-9H-pyrido[2,3-b]indol-9-yl)methyl]phenyl]-N- [ ( 1S ) -2-hydroxy-1-phenylethyl ] ethanamide ( DB04852 ) , a new P55157 inhibitor , to low-density lipoprotein ( LDL ) -receptor-deficient Watanabe heritable hyperlipidemic ( WHHL ) rabbits at doses of 3 , 6 , and 12 mg/kg for 4 weeks . In the 12 mg/kg group , the plasma cholesterol and triglyceride levels were decreased by 70 % and 45 % , respectively , and the very low-density lipoprotein ( VLDL ) secretion rate was decreased by 80 % . The composition of newly secreted VLDL was similar in each group . This suggests that DB04852 diminished the number of VLDL particles secreted from the liver . Although the ratio of vitamin E/LDL was not altered by DB04852 , triglyceride accumulation and a decrease in vitamin E were observed in the liver . In conclusion , an inhibition of VLDL secretion led to a decrease of plasma LDL in WHHL rabbits , and P55157 inhibitors should have hypolipidemic effects against homozygous familial hypercholesterolemia . Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay quantifying olaparib in human plasma . DB09074 is an inhibitor of poly ADP ribose polymerase 1 ( P09874 ) . Phase I and II trials showed promising results of olaparib against tumours in BRCA mutation carriers . Currently an increasing number of clinical trials with olaparib in combination with other compounds or radiotherapy are conducted . To support these clinical trials an LC-MS/MS method was developed and validated for the quantification of olaparib in human plasma . Human plasma samples were collected in the clinic and stored at nominally -20°C . DB09074 was isolated from plasma by liquid-liquid extraction , separated on a C18 column with gradient elution and analyzed with triple quadrupole mass spectrometry in positive ion mode . A deuterated isotope was used as internal standard for the quantification . The assay , ranging from 10 to 5000ng/mL , was linear with correlation coefficients ( r(2) ) of 0.9994 or better . The assay was accurate and precise , with inter-assay and intra-assay accuracies within ±7.6 % of nominal and inter-assay and intra-assay precision ≤9.3 % at the lower limit of quantification and ≤5.7 % at the other concentration levels tested . All results were within the acceptance criteria of the US FDA and the latest P15941 guidelines for method validation . A quantitative method was developed and validated for the quantification of olaparib in human plasma . The method could successfully be applied for the pharmacokinetic quantification of olaparib in cancer patients treated with olaparib . DB05258 receptors are important for antiproliferative effect of interferon-alpha against human hepatocellular carcinoma cells . AIM : Interferon ( IFN ) -alpha is a promising drug for the prevention and treatment of hepatocellular carcinoma ( HCC ) . We reported that responders to IFN-alpha/5-fluorouracil combination therapy expressed higher IFN alpha receptor ( P17181 )2 in tumor . Herein we studied involvement of IFNARs in response to IFN-alpha in HCC cells . METHODS : IFN-alpha sensitivity and expression of IFNARs were studied in six HCC cell lines ( HuH7 , P98160 /PRF/5 , P08246 , HLF , HepG2 , Hep3B ) using growth-inhibitory and RT-PCR , Western blot assays . Short interfering RNAs ( SiRNAs ) against P17181 and 2 were used to analyze the role of the IFNARs in IFN-alpha 's effect and signal transduction . RESULTS : The expressions of P17181 and 2c mRNAs were higher in P98160 /PRF/5 cells than those in other cell lines , and P98160 /PRF/5 cells expressed abundant IFNAR2c on their cell membrane . When we examined the sensitivity of the HCC cell lines to the growth-inhibitory effect of IFN-alpha , P98160 /PRF/5 exhibited a significant response , while the other cells were much more resistant . Knockdown of either P17181 or 2 using siRNAs suppressed the IFN-alpha 's signal transduction ( 2.5-fold ) , and decreased the growth-inhibitory effect ( down by 69.9 % and 67.3 % ) . CONCLUSION : The results suggest that the expression of P17181 and IFNAR2c independently are important for the antiproliferative effect of IFN-alpha in HCC cells . Vascular endothelial growth factor trap-eye and trap technology : DB08885 from bench to bedside . Anti-vascular endothelial growth factor ( P15692 ) currently used to treat eye diseases have included monoclonal antibodies , antibody fragments , and an aptamer . A different method of achieving P15692 blockade in retinal diseases includes the concept of a cytokine trap . Cytokine traps technology are being evaluated for the treatment of various diseases that are driven by excessive cytokine levels . Traps consist of two extracellular cytokine receptor domains fused together to form a human immunoglobulin G ( IgG ) . DB08885 / P15692 trap-eye ( VTE ) is a soluble fusion protein , which combines ligand-binding elements taken from the extracellular components of P15692 receptors 1 and 2 fused to the Fc portion of IgG . This protein contains all human amino acid sequences , which minimizes the potential for immunogenicity in human patients . This review presents the latest data on VTE in regard to the pharmacokinetics , dosage and safety , preclinical and clinical experiences . Method of the literature search : A systematic search of the literature was conducted on PubMed , Scopus , and Google Scholar with no limitation on language or year of publication databases . It was oriented to articles published for VTE in preclinical and clinical studies and was focused on the pharmacokinetics , dosage and safety of VTE . 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 . Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 ( P25098 ) in P29320 -293beta2 cells and cardiac myocytes . Membrane-recruitment of P25098 ( G-protein receptor kinase 2 ) provides a fundamental step in the desensitization process controlling GPCRs ( G-protein-coupled receptors ) , such as the beta2AR ( beta2-adrenergic receptor ) . In the present paper , we show that challenge of P29320 -293beta2 [ human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP ( green fluorescent protein ) ] cells with the beta-adrenoceptor agonist , isoprenaline , causes P25098 to become phosphorylated by PKA ( DB02527 -dependent protein kinase ) . This action is facilitated when DB02527 -specific DB05876 ( phosphodiesterase-4 ) activity is selectively inactivated , either chemically with rolipram or by siRNA ( small interfering RNA ) -mediated knockdown of Q07343 and Q08499 . DB05876 -selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of P25098 , phosphorylation of the beta2AR by P25098 , membrane translocation of beta-arrestin and internalization of beta2ARs . DB05876 -selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of P25098 in cardiac myocytes . In the absence of isoprenaline , rolipram-induced inhibition of DB05876 activity in P29320 -293beta2 cells acts to stimulate PKA phosphorylation of P25098 , with consequential effects on P25098 membrane recruitment and P25098 -mediated phosphorylation of the beta2AR . We propose that a key role for DB05876 enzymes is : ( i ) to gate the action of PKA on P25098 , influencing the rate of P25098 phosphorylation of the beta2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge , and ( ii ) to protect P25098 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of DB02527 .