MH_dev_124

The genes encoding cytokines P60568 REA , P22301 REA and P29460 REA are primary DB00136 SUB target genes . A number of studies have described the effects of DB00136 SUB in immune system . Most of the known effects of DB00136 SUB are indirect since only two functional VDREs that regulate transcription of cytokine gene has been reported until today . In this study we have examined a possibility of direct transcriptional regulation of P60568 REA , P22301 REA and P29460 REA genes in activated Jurkat or THP - 1 cells via liganded P11473 REA by using gene expression analysis and chromatin immunoprecipitation assays . According to our data the P60568 REA , P22301 REA and P29460 REA genes respond to DB00136 SUB treatment by 3-6 h . In addition , all of these genes contain several genomic regions that recruit P11473 REA in a ligand dependent fashion . These data suggest that the above cytokines are under direct transcriptional regulation by DB00136 SUB .

Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase . Human cytochrome P450 4F2 ( P78329 REA ) catalyzes the initial omega-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is , to date , the only enzyme shown to metabolize vitamin E . The objective of this study was to characterize this activity , particularly the influence of key features of tocochromanol substrate structure . The influence of the number and positions of methyl groups on the chromanol ring , and of stereochemistry and saturation of the side chain , were explored using HepG 2 cultures and microsomal reaction systems . Human liver microsomes and microsomes selectively expressing recombinant human P78329 REA exhibited substrate activity patterns similar to those of HepG 2 cells . Although activity was strongly associated with substrate accumulation by cells or microsomes , substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration . Methylation at P01031 REA of the chromanol ring was associated with markedly low activity . Tocotrienols exhibited much higher Vmax values than their tocopherol counterparts . Side chain stereochemistry had no effect on omega-hydroxylation of DB00163 ( alpha-TOH ) by any system . Kinetic analysis of microsomal P78329 REA activity revealed Michaelis-Menten kinetics for alpha-TOH but allosteric cooperativity for other vitamers , especially tocotrienols . Additionally , alpha-TOH was a positive effector of omega-hydroxylation of other vitamers . These results indicate that P78329 REA - mediated tocopherol-omega-hydroxylation is a central feature underlying the different biological half-lives , and therefore biopotencies , of the tocopherols and tocotrienols .

P35367 REA occupancy in human brains after single oral doses of histamine H1 antagonists measured by positron emission tomography . 1 . P35367 REA occupancy in the human brain was measured in 20 healthy young men by positron emission tomography ( PET ) using [ 11C ] - doxepin . 2 . ( + ) - DB01114 MEN , a selective and classical antihistamine , occupied 76.8 + / - 4.2 % of the averaged values of available histamine H1 receptors in the frontal cortex after its administration in a single oral dose of 2 mg . Intravenous administration of 5 mg ( + ) - chlorpheniramine almost completely abolished the binding of [ 11C ] - doxepin to H1 receptors ( H1 receptor occupancy : 98.2 + / - 1.2 % ) . 3 . Terfenadine , a nonsedative antihistamine , occupied 17.2 + / - 14.2 % of the available H1 receptors in the human frontal cortex after its administration in a single oral dose of 60 mg . 4 . There was no correlation between H1 receptor occupancy by terfenadine and the plasma concentration of the active acid metabolite of terfenadine in each subject . 5 . PET data on human brain were essentially compatible with those on H1 receptor occupancy in guinea-pig brain determined by in vivo binding techniques , although for the same H1 receptor occupancy the dose was less in human subjects than in guinea-pigs . 6 . The PET studies demonstrated the usefulness of measuring H1 receptor occupancy with classical and second-generation antihistamines in human brain to estimate their unwanted side effects such as sedation and drowsiness quantitatively .

Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells . The active vitamin D3 metabolite DB00136 SUB ( 1,25 ( OH ) 2D3 ) acts as an antiproliferative and differentiating agent for the monoblastoid cell line U937 and as an important immunologic mediator implicated particularly in the function of cells belonging to the monocyte / macrophage lineage . These effects are controlled by the vitamin D receptor ( P11473 REA ) , a member of the steroid hormone receptor family . The objective of this study was to develop U937 transfectants expressing antisense P11473 REA mRNA , and to use these to examine the role of 1,25 ( OH ) 2D3 - P11473 REA interaction in this lineage . A 2 - kb P11473 REA cDNA insert ( including the complete P11473 REA coding region ) was cloned in an antisense orientation into the EBV episomal vector pMEP 4 under the control of an inducible promoter and transfected into U937 . The resultant cell line , DH42 , was hygromycin resistant , contained P11473 REA cDNA , expressed fewer VDRs than controls , and showed a substantial decrease in antiproliferative response to 1,25 ( OH ) 2D3 . However , 1,25 ( OH ) 2D3 increased the number of cells expressing macrophage cell surface Ags , including P08571 REA and CD11b . A subpopulation of smaller cells did not express the differentiation markers after cadmium stimulation . Cell cycle analysis showed shifts in the distribution of cells from P55008 to S phase , which were more pronounced after cadmium treatment . A considerable proportion of cells were outside the cycle and DNA fragmentation confirmed apoptosis . Thus , the functional outcome of the P11473 REA antisense transfection suggests that in the myelomonocytic lineage , P11473 REA expression may act as a protective mechanism against programmed cell death .

P10275 REA - dependent activation of endothelial nitric oxide synthase in vascular endothelial cells : role of phosphatidylinositol 3 - kinase / akt pathway . The mechanisms of testosterone-induced vasodilatation are not fully understood . This study investigated the effect of testosterone on nitric oxide ( NO ) synthesis and its molecular mechanism using human aortic endothelial cells ( HAEC ) . DB00624 at physiological concentrations ( 1-100 nm ) induced a rapid ( 15-30 min ) increase in NO production , which was associated with phosphorylation and activation of endothelial NO synthase ( P29474 REA ) . Then , the involvement of the androgen receptor ( AR ) , which is abundantly expressed in HAEC , was examined . The effect of testosterone on P29474 REA activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA . In contrast , testosterone-induced P29474 REA phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERalpha small interference RNA . DB02901 MEN , a nonaromatizable androgen , also stimulated P29474 REA phosphorylation . Next , the signaling cascade that leads to P29474 REA phosphorylation was explored . DB00624 stimulated rapid phosphorylation of Akt in a time - and dose-dependent manner , with maximal response at 15-60 min . The rapid phosphorylation of P29474 REA or NO production induced by testosterone was inhibited by Akt inhibitor SH - 5 or by phosphatidylinositol ( PI ) 3 - kinase inhibitor wortmannin . Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p8 5alpha subunit of P19957 REA - kinase . In conclusion , testosterone rapidly induces NO production via AR-dependent activation of P29474 REA in HAEC . Activation of P19957 REA - kinase / Akt signaling and the direct interaction of AR with p8 5alpha are involved , at least in part , in P29474 REA phosphorylation .

Inhibition by 1alpha , 25 - dihydroxyvitamin D3 of activin A-induced differentiation of murine erythroleukemic P12259 REA - 5 cells . DB00136 SUB ( 1alpha , 25 - ( OH ) 2D3 ) and other vitamin D3 ( VD3 ) analogs enhanced the inhibitory effect of Activin A on murine erythroleukemia ( P61006 REA ) cell proliferation and differentiation in a dose-dependent manner . 1alpha , 25 - ( OH ) 2D3 inhibited differentiation more potently than proliferation by one order of magnitude . The VD3 analog study demonstrated either effect of VD3 on P61006 REA cells via vitamin D receptor ( P11473 REA ) , as evidenced from the close relationship with the reported affinities for P11473 REA . The effects of 1alpha , 25 - ( OH ) 2D3 were preceded by the suppression of ornithine decarboxylase ( ODC ) activity , a rate-limiting enzyme in polyamine metabolism . Difluoromethylornithine ( DB06243 ) , an inhibitor of ODC , inhibited P61006 REA cell proliferation , which was reversed by the simultaneous addition of putrescine , a product of ODC , but did not affect differentiation . 1alpha , 25 - ( OH ) 2D3 inhibited cell differentiation during the phenotype-expression stage as reflected by the inhibition of beta-globin gene expression , while it inhibited proliferation in the commitment stage . Furthermore , it seems unlikely that the different effects of VD3 on proliferation and differentiation may be a result of upregulation of P11473 REA or nongenomic action . In summary , it was suggested that 1alpha , 25 - ( OH ) 2D3 inhibited Activin A-induced P61006 REA cell proliferation and differentiation by distinct mechanisms and inhibited the proliferation by inhibiting ODC activity . We demonstrated the presence of 1alpha , 25 - ( OH ) 2D3 action on leukemic cells at physiological concentration , which was distinct from the pharmacological effect of VD3 reported thus far .

Cystatin D is a candidate tumor suppressor gene induced by vitamin D in human colon cancer cells . The active vitamin D metabolite 1alpha , 25 - dihydroxyvitamin D3 [ DB00136 SUB ] has wide but not fully understood antitumor activity . A previous transcriptomic analysis of DB00136 SUB action on human colon cancer cells revealed cystatin D ( P28325 ) , which encodes an inhibitor of several cysteine proteases of the cathepsin family , as a candidate target gene . Here we report that DB00136 SUB induced vitamin D receptor ( P11473 REA ) binding to , and activation of , the P28325 promoter and increased P28325 RNA and protein levels in human colon cancer cells . In cells lacking endogenous cystatin D , ectopic cystatin D expression inhibited both proliferation in vitro and xenograft tumor growth in vivo . Furthermore , cystatin D inhibited migration and anchorage-independent growth , antagonized the Wnt / beta-catenin signaling pathway , and repressed c-MYC expression . Cystatin D repressed expression of the epithelial-mesenchymal transition inducers O95863 REA , O43623 REA , P37275 REA , and O60315 and , conversely , induced P12830 REA and other adhesion proteins . P28325 knockdown using shRNA abrogated the antiproliferative effect of DB00136 SUB , attenuated P12830 REA expression , and increased c-MYC expression . In human colorectal tumors , expression of cystatin D correlated with expression of P11473 REA and P12830 REA , and loss of cystatin D correlated with poor tumor differentiation . Based on these data , we propose that P28325 has tumor suppressor activity that may contribute to the antitumoral action of DB00136 SUB in colon cancer .

Targeting eIF 4GI translation initiation factor affords an attractive therapeutic strategy in multiple myeloma . BACKGROUND : Deregulation of protein synthesis is integral to the malignant phenotype and translation initiation is the rate limiting stage . Therefore , eIF 4F translation initiation complex components are attractive therapeutic targets . METHODS : Protein lysates of myeloma cells ( cell lines / patients ' bone marrow samples ) untreated / treated with bevacizumab were assayed for eIF 4GI expression , regulation ( P15559 REA / proteosome dependent fragmentation ) ( WB , DB00266 MEN , qPCR ) and targets ( WB ) . eIF 4GI was inhibited by knockdown and 4EGI - 1 . Cells were tested for viability ( ELISA ) , death ( FACS ) and eIF 4GI targets ( WB ) . RESULTS : Previously , we have shown that manipulation of P15692 REA in myeloma cells attenuated P06730 REA dependent translation initiation . Here we assessed the significance of eIF 4GI to MM cells . We demonstrated increased expression of eIF 4GI in myeloma cells and its attenuation upon P15692 REA inhibition attributed to elevated P15559 REA / proteasome dependent fragmentation and diminished mRNA levels . Knockdown of eIF 4GI was deleterious to myeloma cells phenotype and expression of specific molecular targets ( Q99717 REA / ERα / HIF 1α / c-Myc ) . Finally , we showed that the small molecule 4EGI - 1 inhibits eIF 4GI and causes a reduction in expression of its molecular targets in myeloma . CONCLUSION : Our findings substantiate that translation initiation of particular targets in MM is contingent on the function of eIF 4GI , critical to cell phenotype , and mark it as a viable target for pharmacological intervention .

Reversal of P-glycoprotein and multidrug-resistance protein-mediated drug resistance in KB cells by 5 - O-benzoylated taxinine K . A newly synthesized taxoid originally from the Japanese yew Taxus cuspidata , 5 - O-benzoylated taxinine K ( Q06187 REA ) was examined for its ability to reverse P-glycoprotein ( P-gp ) and multidrug resistance protein ( MRP ) - mediated multidrug resistance . Q06187 REA reversed the resistance to paclitaxel , doxorubicin ( P35318 REA ) , and vincristine ( VCR ) of KB -8-5 and KB - P06681 REA cells that overexpress P-gp by directly interacting with P-gp . Q06187 REA also moderately reversed the resistance to P35318 REA of KB / MRP cells that overexpress MRP . However , Q06187 REA neither inhibited the transporting activity of MRP nor reduced intracellular glutathione levels in KB / MRP cells . Q06187 REA shifted the distribution of P35318 REA in KB / MRP cells from punctate cytoplasmic compartments to the nucleoplasm and cytoplasm by inhibiting acidification of cytoplasmic organelles . These two functions of Q06187 REA make it able to reverse both P-gp - and MRP-mediated MDR . Q06187 REA in combination with P35318 REA should be useful for treating patients with tumors that overexpress both P-gp and MRP .

The genes of the coactivator Q15596 REA and the corepressor Q9Y618 REA are primary DB00136 SUB targets . The complex of the receptor for the hormone 1alpha , 25 - dihydroxyvitamin D ( 3 ) ( 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) ) , Vitamin D ( 3 ) receptor ( P11473 REA ) , the retinoid X receptor ( RXR ) and a 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) response element ( VDRE ) is considered to be the molecular switch for nuclear 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) signaling . In the presence of ligand the P11473 REA - RXR complex interacts with coactivator ( DB01992 ) proteins that in turn contact components of the basal transcriptional machinery resulting in an enhanced transcription of 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) target genes . In the absence of ligand the P11473 REA remains bound to the DNA and interacts with corepressor ( CoR ) proteins that are involved in gene silencing activity . We treated MCF - 7 breast cancer cells with 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) for increasing amounts of time , extracted mRNA and screened by real-time PCR the members of the P52701 REA DB01992 and NCoR CoR families . We find that of the P52701 REA coactivators , only Q15596 REA was responsive to 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) . Similarly Q9Y618 REA but not NCoR 1 gene transcription was sensitive to 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) treatment . In silico analysis revealed that both Q15596 REA and Q9Y618 REA promoters have substantial numbers of VDREs compared to the promoters of the other family members . These VDREs are formed by direct repeats of the core binding motif RGKTCA with a three nucleotide spacing ( Q93038 REA ) . We suggest that some or all of these Q93038 REA - type VDREs are responsible for the observed responsiveness of Q15596 REA and Q9Y618 REA to 1alpha , 25 ( OH ) ( 2 ) D ( 3 ) .

DB02546 and bortezomib synergistically cause ubiquitinated protein accumulation in prostate cancer cells . PURPOSE : Protein ubiquitination is a novel strategy used to treat malignancies . We investigated whether the histone deacetylase inhibitor vorinostat ( Cayman Chemical , Ann Arbor , Michigan ) and the proteasome inhibitor bortezomib ( LC Laboratories , Woburn , Massachusetts ) would synergistically cause the accumulation of ubiquitinated proteins in prostate cancer cells . MATERIALS AND METHODS : LNCaP , PC - 3 and DU 145 cells ( ATCC ™ ) were treated with vorinostat and / or bortezomib . Cell viability and induction of apoptosis were assessed . In vivo efficacy was evaluated in a murine subcutaneous tumor model using PC - 3 cells . The influence of androgen receptor expression on bortezomib efficacy was examined using RNA interference . Changes in the expression of ubiquitinated proteins , cell cycle associated proteins and acetylated histone were evaluated . RESULTS : P10275 REA expression seemed to decrease bortezomib activity . PC - 3 and DU 145 cells were more susceptible to bortezomib than LNCaP cells and the silencing of androgen receptor expression in LNCaP cells enhanced bortezomib activity . DB02546 and bortezomib synergistically induced apoptosis , inhibited prostate cancer cell growth and suppressed tumor growth in a murine xenograft model . The combination decreased cyclin D1 and cyclin-dependent kinase 4 expression , and increased P38936 REA expression . The combination synergistically caused the accumulation of ubiquitinated proteins and histone acetylation . This histone acetylation was a consequence of the accumulation of ubiquitinated proteins . CONCLUSIONS : DB02546 and bortezomib inhibit the growth of prostate cancer cells synergistically by causing ubiquitinated proteins to accumulate in cells . The current study provides a framework for testing the combination in patients with advanced prostate cancer .

P10275 REA inducing bladder cancer progression by promoting an epithelial-mesenchymal transition . The study investigated the role of androgen receptor ( AR ) as a potential target for the treatment of bladder cancer in regulating epithelial-mesenchymal transition or transformation ( EMT ) . Cell proliferation , and migration capacity were determined in bladder cancer T24 cells treated with small interfering RNA directed against AR , and expression levels of P12830 REA , β-catenin and N - cadherin were assessed using quantitative reverse transcription PCR ( qRT-PCR ) . Tumour cell growth was evaluated in vivo in T24 tumour-bearing nude mice receiving electroporation-assisted administration of anti-AR small interfering RNA . It was found that low AR expression decreased proliferation and migration of bladder cancer cells . In vivo experiments showed that silencing AR expression significantly suppressed AR-positive bladder tumour growth with decreased cell proliferation . Low AR level of T24 bladder cancer cells treated with DB01541 ( DB02901 MEN ) decreased expression of P12830 REA , β-catenin and P19022 REA expression , indicating a strong sensitivity to the EMT and In cells with low AR content , TGF-β induced down-regulation of P12830 REA and β-catenin . It is concluded that suppression of AR expression decreased the production of TGF-β , inhibiting EMT and bladder cancer cell growth in vitro and in vivo , implying that its use might be a potential therapeutic target for the treatment of bladder cancer .

Dacomitinib ( PF - 00299804 ) , an irreversible Pan-HER inhibitor , inhibits proliferation of P04626 REA - amplified breast cancer cell lines resistant to trastuzumab and lapatinib . The human P01133 REA ( HER ) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies . DB00072 MEN and lapatinib are standard treatments for P04626 REA - amplified breast cancer , but a significant number of patients do not respond or develop resistance to these drugs . Here we evaluate the in vitro activity of dacomitinib ( PF - 00299804 ) , an irreversible small molecule pan-HER inhibitor , in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands , and with variable sensitivity to trastuzumab and lapatinib . Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC ( 50 ) values . P04626 REA - amplified lines were far more likely to respond to dacomitinib than nonamplified lines ( RR , 3.39 ; P < 0.0001 ) . Furthermore , P04626 REA mRNA and protein expression were quantitatively associated with response . Dacomitinib reduced the phosphorylation of P04626 REA , P00533 REA , Q15303 REA , AKT , and P29323 REA in the majority of sensitive lines . Dacomitinib exerted its antiproliferative effect through a combined G ( 0 ) - G ( 1 ) arrest and an induction of apoptosis . Dacomitinib inhibited growth in several P04626 REA - amplified lines with de novo and acquired resistance to trastuzumab . Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib . This study identifies P04626 REA - amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in P04626 REA - amplified breast cancers resistant to trastuzumab and lapatinib .

Prolonged treatment with bicalutamide induces androgen receptor overexpression and androgen hypersensitivity . BACKGROUND : Various hormone refractory prostate cancer cell models have been established with androgen depletion and have helped to clarify the mechanism for the transition into androgen-depletion independent status . However , the mechanism of bicalutamide resistance remains unclear because few cell models have been generated . METHODS : We generated a bicalutamide-resistant subline , LNCaP - O43633 REA , from LNCaP after prolonged treatment with bicalutamide . Androgen and / or bicalutamide responsiveness for proliferation and prostate-specific antigen ( PSA ) secretion were examined in vitro and in vivo . DB00624 and dihydrotestosterone ( DB02901 MEN ) levels in xenografted tumors were analyzed by liquid chromatography-tandem mass spectrometry . P10275 REA ( AR ) gene mutation and amplification and AR and pAR ( 210 ) expression were determined . RESULTS : LNCaP - O43633 REA did not grow in an androgen-depleted medium and proliferation was stimulated in a tenfold lower concentration of androgen than that of LNCaP . LNCaP - O43633 REA grew in castrated male mice , and the DB02901 MEN level in grafted LNCaP - O43633 REA tumors was 7.7- fold lower than in LNCaP tumors . DB01128 stimulated LNCaP - O43633 REA proliferation and PSA secretion in vitro and the antitumor activity of bicalutamide against LNCaP - O43633 REA was weaker than that of LNCaP in vivo . Additional AR mutation and AR gene amplification were not detected in LNCaP - O43633 REA , but AR and pAR ( 210 ) expression and PSA secretion in LNCaP - O43633 REA were higher than in LNCaP . CONCLUSIONS : DB01128 - resistant LNCaP - O43633 REA exhibited AR overexpression and hypersensitivity to low levels of androgen . Our data suggests that AR overexpression is a significant mechanism of bicalutamide resistance similar to resistance from chronic androgen depletion . In addition , pAR ( 210 ) overexpression could be a potential mechanism for hypersensitivity to low androgen in LNCaP - O43633 REA .

Q15109 REA / RAGE-mediated autophagy promotes pancreatic tumorigenesis and bioenergetics through the P05231 REA - pSTAT 3 pathway . Pancreatic ductal adenocarcinoma ( PDA ) , the fourth leading cause of cancer death in the United States , is a complex disease that arises in the setting of genetic alterations ( P01116 REA , P38398 REA , Q13485 REA , CDKN 2A / p16 ( INK 4a ) and P04637 REA ) , epigenetic perturbations ( MIR 155 , acetylation and methylation ) and epicellular events ( diabetes and inflammation ) . We have demonstrated that the advanced glycation end product-specific receptor ( Q15109 REA , also called RAGE ) contributes to pancreatic tumorigenesis . Targeted ablation of Q15109 REA diminishes the amount of autophagic flux and attenuates the development of early pancreatic intraepithelial neoplasia ( PanIN ) lesions in a murine model of P01116 REA - drivien carcinogenesis . Autophagy ( programmed cell survival ) , a metabolic process of lysosome-mediated self-digestion , promotes pancreatic cancer growth . In pancreatic tumor cell lines , Q15109 REA - mediated autophagy promotes interleukin - 6 ( P05231 REA ) - induced phosphorylation of signal transducer and activator of transcription 3 ( pSTAT 3 ) and mitochondrial localization of pSTAT 3 . Enhanced mitochondrial pSTAT 3 increases the pool of available DB00171 and increases cellular proliferation . Moreover , we observed a positive feedback loop between activation of autophagy and the P05231 REA - pSTAT 3 pathway , perhaps different from the role of cytosolic nonphosphorylated P40763 REA , which has been reported to inhibit autophagy . These Q15109 REA - dependent changes were found during the earliest stages of pancreatic cancer development . These observations of inflammation and altered metabolism in PDA provide a pathological link to early precursor lesion development . Thus , Q15109 REA is an important inflammatory mediator that modulates crosstalk between prosurvival pathways , P05231 REA - pSTAT 3 and autophagy , in PDA tumor cells , and contributes to early PanIN formation .

Development of an osteoblast / osteoclast co-culture derived by human bone marrow stromal cells and human monocytes for biomaterials testing . The communication of bone-forming osteoblasts and bone-resorbing osteoclasts is a fundamental requirement for balanced bone remodelling . For biomaterial research , development of in vitro models is necessary to investigate this communication . In the present study human bone marrow stromal cells and human monocytes were cultivated in order to differentiate into osteoblasts and osteoclasts , respectively . Finally , a cultivation regime was identified which firstly induces the differentiation of the human bone marrow stromal cells followed by the induction of osteoclastogenesis through the osteoblasts formed - - without the external addition of the factors O14788 REA and P09603 REA . As a feedback on osteoblasts enhanced gene expression of P21815 REA was detected for modifications which facilitated the formation of large multinuclear osteoclasts . Phenotype characterization was performed by biochemical methods ( DNA , LDH , ALP , TRAP 5b ) , gene expression analysis ( ALP , P21815 REA , O14788 REA , P05231 REA , VTNR , P43235 REA , TRAP , Q8IYS5 , P30988 REA ) as well as light microscopy , confocal laser scanning microscopy , and scanning electron microscopy . After establishing this model on polystyrene , similar positive results were obtained for cultivation on a relevant bone substitution material - - a composite xerogel of silica , collagen , and calcium phosphate .

A homozygous inactivating calcium-sensing receptor mutation , Pro 339Thr , is associated with isolated primary hyperparathyroidism : correlation between location of mutations and severity of hypercalcaemia . BACKGROUND : Inactivating mutations of the calcium-sensing receptor ( P41180 REA ) , a G-protein-coupled receptor with extracellular ( O95905 ) , transmembrane ( TMD ) and intracellular ( ICD ) domains , cause familial hypocalciuric hypercalcaemia , neonatal severe primary hyperparathyroidism and occasionally primary hyperparathyroidism in adults . OBJECTIVE : To investigate a patient with typical symptomatic primary hyperparathyroidism for P41180 REA abnormalities . PATIENT AND DESIGN : A 51 - year-old woman with primary hyperparathyroidism was investigated for P41180 REA abnormalities as her severe hypercalcaemia ( 3 · 75 mm ) persisted after the removal of two large parathyroid adenomas and she was the daughter of normocalcaemic consanguineous parents . Following informed consent , P41180 REA mutational analysis was undertaken using leucocyte DNA . Wild-type and mutant P41180 REA constructs were expressed in human embryonic kidney ( P29320 REA ) 293 cells and assessed by measuring their intracellular calcium responses to changes in extracellular calcium . Clinical data were pooled with previous studies to search for genotype-phenotype correlations . RESULTS : The proband was homozygous for a Pro 339Thr P41180 REA missense mutation , located in the O95905 , and her normocalcaemic relatives were heterozygous . The mutant Thr 339 P41180 REA had a rightward shift in its dose-response curve with a significantly higher EC ( 50 ) = 3 · 18 mm ± 0 · 19 compared to the wild-type EC ( 50 ) = 2 · 16 mm ± 0 · 1 ( P < 0 · 01 ) , consistent with a loss-of-function mutation . An analysis of P41180 REA mutations in patients with primary hyperparathyroidism revealed that those of the O95905 were associated with a significantly greater hypercalcaemia that was less likely to be corrected after removal of the parathyroid tumours . CONCLUSIONS : A P41180 REA missense mutation causing a loss-of-receptor-function can cause symptomatic primary hyperparathyroidism in adulthood .

Q06187 REA is a therapeutic target in stem-like cells from multiple myeloma . DB09053 MEN ( Imbruvica ) , a small-drug inhibitor of Q06187 REA ( Q06187 REA ) , is currently undergoing clinical testing in patients with multiple myeloma , yet important questions on the role of Q06187 REA in myeloma biology and treatment are outstanding . Using flow-sorted side population cells from human myeloma cell lines and multiple myeloma primary samples as surrogate for the elusive multiple myeloma stem cell , we found that elevated expression of Q06187 REA in myeloma cells leads to AKT / WNT / β-catenin-dependent upregulation of key stemness genes ( Q01860 REA , P48431 REA , Q9H9S0 REA , and MYC ) and enhanced self-renewal . Enforced transgenic expression of Q06187 REA in myeloma cells increased features of cancer stemness , including clonogenicity and resistance to widely used myeloma drugs , whereas inducible knockdown of Q06187 REA abolished them . Furthermore , overexpression of Q06187 REA in myeloma cells promoted tumor growth in laboratory mice and rendered side population-derived tumors that contained high levels of Q06187 REA more sensitive to the selective , second-generation Q06187 REA inhibitor , CGI 1746 , than side population-derived tumors that harbored low levels of Q06187 REA . Taken together , these findings implicate Q06187 REA as a positive regulator of myeloma stemness and provide additional support for the clinical testing of Q06187 REA - targeted therapies in patients with myeloma .

The biological activities of 1alpha , 25 - dihydroxyvitamin D3 and its synthetic analog 1alpha , 25 - dihydroxy - 16 - ene-vitamin D3 in normal human osteoblastic cells and human osteosarcoma SaOS - 2 cells are modulated by 17 - beta estradiol and dependent on stage of differentiation . We compared the effects of 1alpha , 25 - dihydroxyvitamin D3 [ DB00136 SUB ] and its analog , 1alpha , 25 - dihydroxy - 16 - ene-vitamin D3 [ 1alpha , 25 ( OH ) 2-16- ene-D 3 ] , as well as their interactions with 17 - beta estradiol ( E2 ) on osteoblastic function in our human normal ( HOB ) and osteosarcoma SaOS - 2 cell models representing two different stages of differentiation , the more differentiated HOB + DEX cells and SaOS + DEX cells , and the corresponding less differentiated HOB-DEX and SaOS-DEX cells . The differential effects of DB00136 SUB and 1alpha , 25 ( OH ) 2-16- ene-D 3 and the modulation by E2 on ALP activity in HOB-DEX and HOB + DEX cells were small but significant . The most significant effects were seen in SaOS + DEX cells , in which 1alpha , 25 ( OH ) 2-16- ene-D 3 was 100 - fold more potent than DB00136 SUB , the maximal enhancement being exerted at 0.1 nM and 10 nM , respectively . E2 enhanced the stimulatory effects of both compounds , with ALP being increased 2 - fold at 0.1 nM ( p < 0.001 ) . P02818 REA ( OC ) production in HOB-DEX cells was stimulated 1.3 to 1.4- fold by DB00136 SUB and 1alpha , 25 ( OH ) 2-16- ene-D 3 at a concentration of 0.01 nM , with E2 inhibiting the effect of 1alpha , 25 ( OH ) 2-16- ene-D 3 . In SaOS-DEX and SaOS + DEX cells , DB00136 SUB and 1alpha , 25 ( OH ) 2-16- ene-D 3 stimulated OC production 1.6- fold at 0.1 nM with E2 slightly enhancing the effect of DB00136 SUB . Western blot analysis of DB00136 SUB receptor ( P11473 REA ) levels showed that in SaOS + DEX cells , the effect of DB00136 SUB was larger than that of 1alpha , 25 ( OH ) 2-16- ene-D 3 . These results show that 1alpha , 25 ( OH ) 2-16- ene-D 3 is biologically active in human osteoblasts .

Influence of cholecalciferol supplementation in hemodialysis patients on monocyte subsets : a randomized , double-blind , placebo-controlled clinical trial . BACKGROUND / AIMS : Although most hemodialysis patients share a significant 25 - hydroxyvitamin D [ 25 ( OH ) D ] deficiency , supplementation is controversially discussed . A potential influence on monocyte and T lymphocyte dysfunction advocates blood level-adapted supplementation as recommended by K / DOQI guidelines . This was a prospective double-blind randomized placebo controlled trial examining immune effects of 12 weeks of cholecalciferol supplementation . METHODS : We initiated serum level-adapted de novo cholecalciferol supplementation in 38 hemodialysis patients . Outcome measures were : monocyte subset cell counts ( P08571 REA + CD16 + + vs . P08571 REA + + CD16 + vs . P08571 REA + + CD16 - ) , lymphocyte Th1 / Th2 differentiation frequencies , serum inflammatory proteins CRP and TNFα , parathyroid hormone ( PTH ) , Q9GZV9 , and α - Q9UEF7 . RESULTS : At baseline , the mean 25 ( OH ) D serum level in the study population was 31.7 ± 14.3 nmol / l , and only 3 % of patients had levels within the normal range . At 12 weeks , 25 ( OH ) D levels were normalized in the verum group ( 87.8 ± 22.3 vs . placebo 24.6 ± 8.0 nmol / l , p < 0.0001 ) . In parallel , 1,25 ( OH ) 2D levels increased in the verum group . Monocyte subset cell counts as well as Th1 and Th2 lymphocyte frequencies did not change significantly after 12 weeks of cholecalciferol supplementation . There was also no significant difference in PTH , alkaline phosphatase , calcium , phosphate , TNFα , Q9GZV9 , α - Q9UEF7 and CRP levels . CONCLUSIONS : Oral cholecalciferol supplementation according to the K / DOQI recommendations normalizes 25 ( OH ) D levels without relevant side effects such as hyperphosphatemia or hypercalcemia . However , beneficial pleiotropic effects on monocyte subset cell counts , T cell differentiation , or cytokine production could not be confirmed at least at the used dosage . PTH and Q9GZV9 levels were not affected during cholecalciferol administration .

[ Gene polymorphism of the vitamin D receptor , vitamin D-binding protein and calcium-sensing receptor in respect of calcium-phosphate disturbances in chronic dialysis patients ] . Dialysed patients suffering from chronic kidney disease ( CKD ) show varied levels of concentration of parathyroid hormone ( PTH ) in the blood . One of the factors in charge of regulating levels of PTH concentration is DB00136 SUB [ 1,25- ( OH ) 2D3 ] . Its deficiency in advanced stages of CKD is common . Vitamin D supplementation is not always effective in reaching levels of PTH concentration recommended by KDIGO for the dialysed patients . That suggests , among other things , disturbances in 1,25- ( OH ) 2D3 , reaching its place of target effect and having the desired final result . Disturbances of vitamin D target pathway can be genetically conditioned , hence the aim of this paper is to describe the distribution of polymorphic variants of vitamin D-binding protein gene ( VDBP ) , vitamin D receptor gene ( P11473 REA ) and gene of the calcium-sensing receptor ( P41180 REA ) with respect to PTH concentrations in serum and response to cinacalcet treatment in patients with secondary hyperparathyroidism in view of the differences in demographical , clinical and laboratory data of the dialysed patients .

Anti-inflammatory secondary metabolites from the leaves of Rosa laevigata . Bioassay-guided fractionation of a n-BuOH-soluble extract of the leaves of Rosa laevigata led to the isolation of three new 19 - oxo -18,19- seco-ur-sane-type triterpenoids , laevigins A-C ( 1-3 ) , a new oleanane-type triterpenoid saponin , laevigin D ( 4 ) , a new geranylmethylbenzoate , 5 - [ ( 2 ″ E , 6 ″ S ) - 6 ″ , 7 ″ - dihydroxy - 3 ″ , 7 ″ - dimethyl - 2 ″ - octen - 1 ″ - yl ] - 2 - ( β-D-glucopyranosyloxy ) - methyl benzoate ( 5 ) , together with 9 known compounds ( 6-14 ) . Their structures were elucidated by spectroscopic and chemical methods . Compounds 4 , 9 , 11 , and 12 significantly suppressed the LPS-stimulated NF-κB transcriptional activity and the release of TNFα , IL - 1β , P05231 REA , and P22301 REA in mouse RAW 264.7 macrophages . The compound 12 exhibited moderate inhibition on NF-κB transcriptional activity with an IC50 value of 23.21 μM . The IC50 values of compound 12 were measured as 14.32 , 8.53 , 8.04 , and 10.38 μM for the inhibitory activity on TNFα-release , IL - 1β - release , P05231 REA - release , and P22301 REA - release , respectively .

Release of cytokines by blood monocytes during strenuous exercise . During strenuous exercise in endurance athletes , monocytes are activated and there is an acute inflammation and hypoxemia possibly due to lesional pulmonary edema . P05231 REA and P01375 REA released by monocytes may be implicated in the acute phase of lesional pulmonary edema . A study was carried out to determine whether P01375 REA and P05231 REA are released during strenuous exercise , and , if adrenalin released during exercise alters their generation . Ten young and six master athletes underwent an incremental exercise test . Arterial blood was drawn at rest , at the end of the exercise , and 20 minutes afterwards . Monocytes were isolated and incubated for 18 hours in the presence or absence of adrenalin . Il - 6 and P01375 REA were measured in monocyte supernatants . The spontaneous release of P05231 REA or P01375 REA was increased in young athletes when compared to older subjects . The spontaneous release of P01375 REA was increased , but not significantly , by exercise and there was no correlation between the release of P05231 REA and P01375 REA and lung function measured during hypoxemia . DB00668 MEN inhibited the release of P05231 REA or P01375 REA . Correlations were observed between the in vitro release of P05231 REA or P01375 REA and age , VO2max , maximal ventilation and maximal power output of the subjects .

Differential radiosensitisation by ZD1839 ( DB00317 MEN ) , a highly selective epidermal growth factor receptor tyrosine kinase inhibitor in two related bladder cancer cell lines . The epidermal growth factor receptor ( P00533 REA ) is expressed in a wide variety of epithelial tumours including carcinoma of the bladder . Stimulation of the P00533 REA pathway is blocked by ZD1839 ( DB00317 MEN ) , a highly selective P00533 REA tyrosine kinase inhibitor . Radical radiotherapy is an established organ sparing treatment option for muscle invasive bladder cancer and this study has explored the possibility for the use of ZD1839 as a radiosensitiser in this scenario . The effect of combination treatment with ZD1839 ( 0.01 microM ) and ionising radiation in the established bladder cancer cell lines MGH-U 1 and its radiosensitive mutant clone S40b was measured by clonogenic assays . A highly significant radiosensitising effect was seen in both cell lines ( P < 0.001 for MGH-U 1 and S40b cell lines ) . This effect was independent of the concentration of the drug and the duration of exposure prior to treatment with ionising radiation . Cell cycle kinetics of both cell lines was not significantly altered with ZD1839 ( 0.01 microM ) as a single agent . A modest induction of apoptosis was observed with ZD1839 ( 0.01 microM ) as a single agent , but a marked induction was observed with the combination treatment of ZD1839 and ionising radiation . These results suggest a potentially important role for ZD1839 in combination with radiotherapy in the treatment of muscle invasive bladder cancer .

First report of warfarin dose requirements in patients possessing the P11712 REA * 12 allele . BACKGROUND : DB00682 MEN is the most frequently prescribed anticoagulant in North America and Europe . It is administered as a racemate , but S-warfarin is principally responsible for its anticoagulant activity . Cytochrome P450 ( CYP ) 2C9 is the enzyme primarily responsible for the metabolism of S-warfarin . Numerous variant alleles of P11712 REA have been identified . The P11712 REA * 12 ( rs9332239 ) allele harbors a P489S substitution in P11712 REA which has been shown to result in a 40 % decline in catalytic activity in vitro . CASES : Four Caucasian patients with a low mean weekly warfarin dose ( MWWD ) were genotyped for P11712 REA , Q9BQB6 and P02649 REA variant alleles . None of the four patients carried the common P11712 REA variant alleles ( * 2 , * 3 , * 5 , * 6 , * 7 , * 8 , * 9 , * 11 , * 13 ) despite a relatively low MWWD ( 23.4 ± 7.94 mg ) compared to 208 patients carrying the CYP 29C9 * 1 genotype ( 32.2 ± 12.65 mg ) . Given that P11712 REA * 12 confers decreased in vitro activity to the enzyme , we investigated whether these patients carried this allele . All four patients were P11712 REA * 12 CT heterozygotes . Individual comparisons with patients possessing the same Q9BQB6 and P02649 REA genotypes also demonstrated lower dose requirements in the patients that possessed P11712 REA * 12 allele . CONCLUSIONS : There are no reports of the clinical impact of rs9332239 on P11712 REA substrates . This is the first report of patients with the rare P11712 REA * 12 genotype and lower warfarin dose requirements .

A concise and efficient route to 2alpha - ( omega-hydroxyalkoxy ) - 1alpha , 25 - dihydroxyvi tam in D3 : remarkably high affinity to vitamin D receptor . [ reaction : see text ] A convenient and potentially valuable synthetic approach to the novel 2alpha - functionalized 1alpha , 25 - dihydroxyvitamin D3 [ DB00136 SUB ] derivatives ( 1a - c ) , which are the P06681 REA - epimer of ED - 71 and its analogues , has been developed . The C2alpha - modified ring A precursors ( 1,7- enynes 16 , n = 0 , 1 , and 2 ) were constructed stereoselectively starting from D-glucose in high yield . In the synthesized 2alpha - ( omega-hydroxyalkoxy ) - DB00136 SUB derivatives , 1a and 1b showed a greater binding affinity to vitamin D receptor ( P11473 REA ) , up to 1.8 times that of the native hormone .

1,25- Dihydroxycholecalciferol enhances butyrate-induced P38936 REA ( Waf 1 / Cip 1 ) expression . Butyrate , a short-chain fatty acid produced in the colon , as well as its prodrug tributyrin , reduce proliferation and increase differentiation of colon cancer cells . P38936 REA ( Waf 1 / Cip 1 ) and p27 ( Kip 1 ) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines . We studied the effects of butyrate on differentiation , P11473 REA expression , as well as on P38936 REA ( Waf 1 / Cip 1 ) and p27 ( Kip 1 ) expression in human colon cancer cells ( Caco - 2 ) . Butyrate induced cell differentiation , which was further enhanced after addition of DB00136 SUB . Synergistic effect of butyrate and dihydroxycholecalciferol in Caco - 2 cells was due to butyrate-induced overexpression of P11473 REA . While butyrate as well as dihydroxycholecalciferol increased P38936 REA ( Waf 1 / Cip 1 ) and p27 ( Kip 1 ) expression , in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of P38936 REA ( Waf 1 / Cip 1 ) , but not of p27 ( Kip 1 ) expression . These data imply that butyrate selectively increases P38936 REA ( Waf 1 / Cip 1 ) expression via upregulation of P11473 REA in Caco - 2 cells .

DB00136 SUB induces nitric oxide production in cultured endothelial cells . BACKGROUND : Recently , DB00136 SUB ( vitD ) has received increasing interest for its effects on many tissues and organs other than bone . A number of experimental studies have shown that vitD may have an important role in modifying risk for cardiovascular disease . AIMS : This study was planned to test the effects of vitD on endothelial nitric oxide ( NO ) production and to study the intracellular pathways leading to NO release . METHODS : In human umbilical vein endothelial cells ( HUVEC ) cultures the effects of vitD on NO production and p38 , Akt , P29323 REA and P29474 REA phosphorylations were examined in absence or in presence of the NO synthase inhibitor L-NAME and protein kinases specific inhibitors SB203580 , wortmannin and UO126 . RESULTS : VitD caused a concentration-dependent increase in NO production . The maximum effect was observed at a concentration of 1 nM and the optimal time of stimulation was 1 min . Effects induced by vitD were abolished by L-NAME and by pre-treatment with protein kinases inhibitors . To verify the effective involvement of vitD receptor ( P11473 REA ) in the action mechanism of vitD , experiments were repeated in presence of the specific P11473 REA ligands ZK159222 and ZK191784 . CONCLUSIONS : The results of this study demonstrate that vitD can induce a significant increase in endothelial NO production . VitD interaction with P11473 REA caused the phosphorylation of p38 , AKT and P29323 REA leading to P29474 REA activation .

c-Fos protein as a target of anti-osteoclastogenic action of vitamin D , and synthesis of new analogs . Although active vitamin D drugs have been used for the treatment of osteoporosis , how the vitamin D receptor ( P11473 REA ) regulates bone cell function remains largely unknown . Using osteoprotegerin-deficient mice , which exhibit severe osteoporosis due to excessive receptor activator of NF-kappaB ligand / receptor activator of NF-kappaB ( O14788 REA / Q9Y6Q6 REA ) stimulation , we show herein that oral treatment of these mice with 1alpha , 25 - dihydroxyvitamin D3 [ DB00136 SUB ] inhibited bone resorption and prevented bone loss , suggesting that P11473 REA counters O14788 REA / Q9Y6Q6 REA signaling . In P09603 REA - dependent osteoclast precursor cells isolated from mouse bone marrow , DB00136 SUB potently and dose-dependently inhibited their differentiation into multinucleate osteoclasts induced by O14788 REA . Among signaling molecules downstream of Q9Y6Q6 REA , DB00136 SUB inhibited the induction of c-Fos protein after O14788 REA stimulation , and retroviral expression of c-Fos protein abrogated the suppressive effect of DB00136 SUB on osteoclast development . By screening vitamin D analogs based on their c-Fos-suppressing activity , we identified a new analog , named DD281 , that inhibited bone resorption and prevented bone loss in ovariectomized mice , more potently than DB00136 SUB , with similar levels of calcium absorption . Thus , c-Fos protein is an important target of the skeletal action of P11473 REA - based drugs , and DD281 is a bone-selective analog that may be useful for the treatment of bone diseases with excessive osteoclastic activity .

Progenitor cells harvested from bovine follicles become endothelial cells . Hematopoietic-like colonies develop in post-confluent granulosa cell cultures derived from bovine antral follicles . Previously , we had shown that these colonies gave rise to macrophages . In the present study , we validated the presence of somatic P10721 REA - positive ( P10721 REA ( + ) ) progenitor cells in colony-containing granulosa cell cultures . The cultures expressed the progenitor cell markers Sox - 2 , Oct 3/4 , P10721 REA , and alkaline phosphatase in western blot analysis . The successful double immunofluorescence localization of P10721 REA and P08571 REA , P08575 REA , CD133 , or P15692 REA - R2 revealed a specific subpopulation of progenitor cells . Flow cytometry showed that cells doubly positive for P10721 REA and P08571 REA or P08575 REA comprised less than 10 % of the population . The P10721 REA ( + ) cells were purified by magnetic selection and differentiated with the hanging drop technique using haematopoietic differentiation medium . Pure cultures of either granulosa cells or endothelial cells were obtained . The spindle-shaped and epithelioid phenotypes indicated endothelial cell heterogeneity of microvascular source . We conclude that progenitor cells are obtained from the follicle harvest , which differentiate into endothelial cells . The cells are relevant for findings to angiogenesis and luteinization of the corpus luteum .

The human peroxisome proliferator-activated receptor delta gene is a primary target of 1alpha , 25 - dihydroxyvitamin D3 and its nuclear receptor . Peroxisome proliferator-activated receptor ( Q07869 REA ) delta is the most widely expressed member of the Q07869 REA family of nuclear receptor fatty acid sensors . Real-time PCR analysis of breast and prostate cancer cell lines demonstrated that PPARdelta expression was increased 1.5 to 3.2- fold after three hours stimulation with the natural vitamin D receptor ( P11473 REA ) agonist , 1alpha , 25 - dihydroxyvitamin D3 ( DB00136 SUB ) . In silico analysis of the 20 kb of the human PPARdelta promoter revealed a Q93038 REA - type DB00136 SUB response element approximately 350 bp upstream of the transcription start site , which was able to bind P11473 REA - retinoid X receptor ( RXR ) heterodimers and mediate a DB00136 SUB - dependent upregulation of reporter gene activity . Chromatin immuno-precipitation assays demonstrated that a number of proteins representative for DB00136 SUB - mediated gene activation , such as P11473 REA , RXR and RNA polymerase II , displayed a DB00136 SUB - dependent association with a region of the proximal PPARdelta promoter that contained the putative Q93038 REA - type VDRE . This was also true for other proteins that are involved in or are the subject of chromatin modification , such as the histone acetyltransferase CBP and histone 4 , which displayed ligand-dependent association and acetylation , respectively . Finally , real-time PCR analysis demonstrated that DB00136 SUB and the synthetic PPARdelta ligand L783483 show a cell and time-dependent interference in each other ' s effects on P11473 REA mRNA expression , so that their combined application shows complex effects on the induction of P11473 REA target genes , such as Q07973 REA . Taken together , we conclude that PPARdelta is a primary DB00136 SUB - responding gene and that P11473 REA and PPARdelta signaling pathways are interconnected at the level of cross-regulation of their respective transcription factor mRNA levels .

Genome-wide association study identifies genetic determinants of warfarin responsiveness for Japanese . DB00682 MENMAX DB00682 MEN is a commonly used anticoagulant , whose dose needs to be determined for each individual patient owing to large inter-individual variability in its therapeutic dose . Although several clinical and genetic variables influencing warfarin dose have been identified , uncovering additional factors are critically important for safer use of warfarin . Through a genome-wide association study , we identified single-nucleotide polymorphism ( SNP ) rs2108622 [ cytochrome P450 , family 4 , subfamily F , polypeptide 2 ( P78329 REA ) ] as a genetic determinant of warfarin responsiveness for Japanese . Stratifying subjects who have been pre-classified according to the genotypes of SNP rs10509680 [ cytochrome P450 , family 2 , subfamily C , polypeptide 9 ( P11712 REA ) ] and SNP rs9923231 [ vitamin K epoxide reductase complex subunit 1 ( Q9BQB6 ) ] , based on their genotypes of rs2108622 allowed identification of subjects who require higher dose of warfarin . Incorporating genotypes of rs2108622 into a warfarin dosing algorithm that considers age , body surface area , status of amiodarone co-administration and genotypes of SNPs in the P11712 REA and Q9BQB6 genes improved the model ' s predictability to 43.4 % . In this study , the association of P78329 REA with warfarin dose of the Japanese has been established for the first time . Besides , a warfarin dosing algorithm that incorporates genotypes of rs2108622 and amiodarone co-administration status was suggested for the Japanese . Our study also implied that common SNPs other than those in the P11712 REA , Q9BQB6 and P78329 REA genes that show strong effect on the therapeutic warfarin dose might not exist .

Autosomal-dominant hypophosphatemic rickets ( P30518 REA ) mutations stabilize Q9GZV9 . BACKGROUND : The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets ( P30518 REA ) is Q9GZV9 , which encodes a secreted protein related to the fibroblast growth factors ( FGFs ) . We previously detected missense mutations R176Q , R179W , and R179Q in Q9GZV9 from P30518 REA kindreds . The mutations replace R residues within a subtilisin-like proprotein convertase ( Q969E3 REA ) cleavage site 176RHTR - 179 ( RXXR motif ) . The goal of these studies was to determine if the P30518 REA mutations lead to protease resistance of Q9GZV9 . METHODS : The P30518 REA mutations were introduced into human Q9GZV9 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK 293 cells . Protein expression was determined by Western analyses . RESULTS : Antibodies directed toward the C-terminal portion of Q9GZV9 revealed that the native Q9GZV9 protein resolved as 32 kD and 12 kD species in HEK 293 conditioned media ; however , the three mutated proteins were detected only as the 32 kD band . An N-terminal FLAG-tagged native Q9GZV9 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody , whereas the R176Q mutant resolved primarily as the 36 kD protein species . Cleavage of Q9GZV9 was not enhanced by extracellular incubation of Q9GZV9 with HEK 293 cells . Native and mutant FGF - 23s bound heparin . CONCLUSIONS : Q9GZV9 proteins containing the P30518 REA mutations are secreted , and produce polypeptides less sensitive to protease cleavage than wild-type Q9GZV9 . Therefore , the P30518 REA mutations may protect Q9GZV9 from proteolysis , thereby potentially elevating circulating concentrations of Q9GZV9 and leading to phosphate wasting in P30518 REA patients .

Antagonistic action of novel 1alpha , 25 - dihydroxyvitamin D3 - 26 , 23 - lactone analogs on differentiation of human leukemia cells ( HL - 60 ) induced by 1alpha , 25 - dihydroxyvitamin D3 . We examined the effects of two novel 1alpha , 25 - dihydroxyvitamin D3 -26,23- lactone ( 1alpha , 25 - lactone ) analogues on human promyelocytic leukemia cell ( HL - 60 ) differentiation using the evaluation system of the vitamin D nuclear receptor ( P11473 REA ) / vitamin D-responsive element ( DRE ) - mediated genomic action stimulated by 1alpha , 25 - dihydroxyvitamin D3 ( DB00136 SUB ) and its analogues . We found that the 1alpha , 25 - lactone analogues ( 23S ) - 25 - dehydro - 1alpha - hydroxyvitamin-D 3-26 , 23 - lactone ( TEI - 9647 ) , and ( 23R ) - 25 - dehydro - 1alpha - hydroxyvitamin-D 3-26 , 23 - lactone ( TEI - 9648 ) bound much more strongly to the P11473 REA than the natural ( 23S , 25R ) - DB00136 SUB -26,23- lactone , but did not induce cell differentiation even at high concentrations ( 10 ( - 6 ) M ) . Intriguingly , the differentiation of HL - 60 cells induced by DB00136 SUB was inhibited by either TEI - 9647 or TEI - 9648 but not by the natural lactone . In contrast , retinoic acid or 12 - O-tetradecanoylphorbol - 13 - acetate-induced HL - 60 cell differentiation was not blocked by TEI - 9647 or TEI - 9648 . In separate studies , TEI - 9647 ( 10 ( - 7 ) M ) was found to be an effective antagonist of both DB00136 SUB ( 10 (-8 ) M ) mediated induction of P38936 REA ( P38936 REA , CIP 1 ) in HL - 60 cells and activation of the luciferase reporter assay in COS - 7 cells transfected with cDNA containing the DRE of the rat DB00146 - 24 - hydroxylase gene and cDNA of the human P11473 REA . Collectively the results strongly suggest that our novel 1alpha , 25 - lactone analogues , TEI - 9647 and TEI - 9648 , are specific antagonists of 1alpha , 25 ( OH ) 2D3 action , specifically P11473 REA / DRE-mediated genomic action . As such , they represent the first examples of antagonists , which act on the nuclear P11473 REA .

Peroxisome proliferator-activated receptor-gamma agonists increase vascular endothelial growth factor expression in human vascular smooth muscle cells . Vascular endothelial growth factor ( P15692 REA ) , expressed in a variety of mesenchymal cells including vascular smooth muscle cells ( VSMC ) , is a potent mitogen for endothelial cells , and is used clinically applied for ischemic disease of peripheral vessels . To determine whether peroxisome proliferator-activated receptor gamma ( PPARgamma ) regulates P15692 REA production in VSMC , we examined P15692 REA secretion from VSMC treated with Q07869 REA agonists . DB00197 increased P15692 REA secretion in a time - and dose-dependent manner ( 261 + / - 35 % with 25 mM of troglitazone for 24 h ) , and also increased levels of P15692 REA mRNA . P15692 REA secretion was also increased by other PPARgamma agonists , pioglitazone , LY171883 , and 15d - PGJ 2 ( 224 + / - 17.1 % , 247 + / - 36.8 % and 171 + / - 7.8 % , respectively ) , but not the PPARgamma agonists bezafibrate and Wy14643 ( 85.2 + / - 1.5 % , 94.6 + / - 3.2 , respectively ) . Our findings suggest that thiazolidinediones might be useful for the therapeutic angiogenesis for ischemic artery disease .

Role of vitamin D receptor in the antiproliferative effects of calcitriol in tumor-derived endothelial cells and tumor angiogenesis in vivo . Calcitriol ( DB00136 SUB ) , the major active form of vitamin D , is antiproliferative in tumor cells and tumor-derived endothelial cells ( TDEC ) . These actions of calcitriol are mediated at least in part by vitamin D receptor ( P11473 REA ) , which is expressed in many tissues including endothelial cells . To investigate the role of P11473 REA in calcitriol effects on tumor vasculature , we established TRAMP - 2 tumors subcutaneously into either P11473 REA wild-type ( WT ) or knockout ( KO ) mice . Within 30 days post-inoculation , tumors in KO mice were larger than those in WT ( P < 0.001 ) . TDEC from WT expressed P11473 REA and were able to transactivate a reporter gene whereas TDEC from KO mice were not . Treatment with calcitriol resulted in growth inhibition in TDEC expressing P11473 REA . However , TDEC from KO mice were relatively resistant , suggesting that calcitriol-mediated growth inhibition on TDEC is P11473 REA - dependent . Further analysis of the TRAMP - P06681 REA tumor sections revealed that the vessels in KO mice were enlarged and had less pericyte coverage compared with WT ( P < 0.001 ) . Contrast-enhanced magnetic resonance imaging showed an increase in vascular volume of TRAMP tumors grown in P11473 REA KO mice compared with WT mice ( P < 0.001 ) and FITC-dextran permeability assay suggested a higher extent of vascular leakage in tumors from KO mice . Using ELISA and Western blot analysis , there was an increase of hypoxia-inducible factor - 1alpha , vascular endothelial growth factor , angiopoietin 1 , and platelet-derived growth factor-BB levels observed in tumors from KO mice . These results indicate that calcitriol-mediated antiproliferative effects on TDEC are P11473 REA - dependent and loss of P11473 REA can lead to abnormal tumor angiogenesis .

Maximizing clinical benefit with trastuzumab . To optimize patient management in breast cancer a number of factors are considered , including hormone receptor and P04626 REA status . A feasible approach for women with less aggressive , estrogen receptor / P04626 REA - positive metastatic breast cancer is to consider trastuzumab ( Herceptin ; F . Hoffmann-La Roche , Basel , Switzerland ) combined with endocrine therapy . Randomized clinical trials are ongoing to assess the combination of trastuzumab with aromatase inhibitors . In patients with aggressive P04626 REA - positive metastatic breast cancer , trastuzumab / chemotherapy combination regimens are warranted . When administered first line in combination with a taxane , trastuzumab improves all clinical outcome parameters , including survival , in such patients . DB00072 MEN adds little to the toxicity profile of taxanes , and trastuzumab combination therapy is associated with improvements in quality of life when compared with chemotherapy alone . There is encouraging evidence of improved efficacy when trastuzumab is combined with other cytotoxic agents with proven single-agent activity in breast cancer , including capecitabine ( DB01101 ; F . Hoffmann-La Roche ) , gemcitabine , and vinorelbine . DB00072 MEN is also being investigated as part of triplet drug regimens . DB00072 MEN has good single-agent activity in first-line therapy . This is of relevance to women with P04626 REA - positive disease who are not suitable for , or do not wish to receive , cytotoxic chemotherapy . The benefits noted with trastuzumab-containing regimens were documented in clinical trials where trastuzumab was given until disease progression . A further rationale exists to continue trastuzumab beyond progression . Data from retrospective reviews indicate that this strategy is feasible .

P00533 REA expression and suramin cytotoxicity in vitro . Twenty-five cell lines derived from nine different human cancers were tested for the cytotoxic activity of suramin . Two different initial cellular concentrations were used : C1 ( 800-2000 cells per well ) and P06681 REA ( 3000-7000 cells per well ) . Suramin concentrations ranged from 50 to 2500 micrograms / ml . Cytotoxicity was assessed by the MTT test . Epidermal growth factor receptors ( P00533 REA ) were assayed by competition analysis and Scatchard plots . In sixteen cell lines suramin had an unexpected growth stimulation effect at low concentration ( 50-125 micrograms / ml ) . IC50 varied from 21 micrograms / ml ( osteosarcoma , OS2 ) to 1408 micrograms / ml ( melanoma , Q9HD26 24 ) and , within melanoma cell lines , it varied from 120 micrograms / ml ( Q9HD26 41 ) to 1408 micrograms / ml ( Q9HD26 24 ) . The individual IC50 values were positively and significantly linked with the initial cellular density . Eighteen cell lines had measurable P00533 REA ( six with two families of sites , twelve with one ) : Kd varied between 0.004 nmol / l for the highest affinity site ( melanoma , Q9HD26 7 ) to 1.852 nmol / l for the lowest affinity site ( lung , Q9HD26 12 ) . There was no relation between presence or absence of P01133 REA binding sites and distribution of IC50 , but for cells with measurable P00533 REA there was a weak but significant correlation between the number of P01133 REA binding sites per cell and the corresponding IC50 ( r = -0.53 , P = 0.021 ) .

Endotoxin induces proliferation of NSCLC in vitro and in vivo : role of P35354 REA and P00533 REA activation . Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease . Therefore , we investigated the effect of bacterial lipopolysaccharides ( LPS ) on tumor proliferation in vitro in the non-small cell lung cancer ( NSCLC ) cell line A549 , ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model . LPS induced a time - and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting . In parallel , an increased expression of the proliferation marker Ki - 67 and cyclooxygenase ( P36551 REA ) - 2 was detected both in A549 cells and in ex vivo human NSCLC tissue . Large amounts of P35354 REA - derived prostaglandin ( PG ) E ( 2 ) were secreted from LPS-stimulated A549 cells . Pharmacological interventions revealed that the proliferative effect of LPS was dependent on P08571 REA and Toll-like receptor ( TLR ) 4 . Moreover , blocking of the epidermal growth factor receptor ( P00533 REA ) also decreased LPS-induced proliferation of A549 cells . Inhibition of P35354 REA activity in A549 cells severely attenuated both PGE ( 2 ) release and proliferation in response to LPS . Synthesis of PGE ( 2 ) was also reduced by inhibiting P08571 REA , O00206 REA and P00533 REA in A549 cells . The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki - 67 expression in implanted tumors . In summary , LPS induces proliferation of NSCLC cells in vitro , ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC . Pulmonary infection may thus directly induce tumor progression in NSCLC .

Inhibition of serum-stimulated mitogen activated protein kinase by 1alpha , 25 ( OH ) 2 - vitamin D3 in MCF - 7 breast cancer cells . DB00136 SUB [ DB00136 SUB ] , the hormonally active form of vitamin D3 , has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo . DB00136 SUB acts through two different mechanisms . In addition to regulating gene transcription via its specific intracellular receptor ( vitamin D receptor , P11473 REA ) , DB00136 SUB induces rapid , non-transcriptional responses involving activation of transmembrane signal transduction pathways , like growth factors and peptide hormones . The mechanisms that mediate the antiproliferative effects of DB00136 SUB in breast cancer cells are not fully understood . Particularly , there is no information about the early non-genomic signal transduction effectors modulated by the hormone . The present study shows that DB00136 SUB rapidly inhibits serum induced activation of P27361 REA and P28482 REA Q96HU1 kinases . The tyrosine kinase Src is involved in the pathway leading to activation of P29323 REA 1/2 by serum . Furthermore , DB00136 SUB increases the tyrosine-phosphorylated state of Src and inhibits its kinase activity , while induces the association of the P11473 REA with Src , either in the presence or absence of serum . In parallel , the hormone rapidly increases the amounts of P11473 REA associated to plasma membranes ( PM ) . Pretreatment with the tyrosine phosphatase inhibitors orthovanadate or bpV ( phen ) prevented mitogen-activated protein kinase ( MAPK ) inhibition by DB00136 SUB . These data altogether suggest that DB00136 SUB inhibits the MAPK cascade by inactivating Src tyrosine kinase through a mechanism mediated by the P11473 REA and tyrosine phosphatases .

Q13507 REA - like protein and vitamin D receptor mediate DB00136 SUB - induced Q5T124 influx in muscle cells . 1alpha , 25 - Dihydroxy-Vitamin-D 3 ( 1alpha , 25 ( OH ) 2 - DB00169 ) stimulates in skeletal muscle cells Ca2 + release from inner stores and influx through both voltage-dependent and store-operated Ca2 + ( Q5T124 , CCE ) channels . We investigated the involvement of TRPC proteins and Vitamin D receptor ( P11473 REA ) in CCE induced by DB00136 SUB in chick muscle cells . Two fragments were amplified by RT-PCR , exhibiting approximately 80 % sequence homology with mammalian Q13507 REA / 6/7 . Northern and Western blots employing a Q13507 REA - probe and anti - Q13507 REA antibodies , respectively , confirmed endogenous expression of a Q13507 REA - like protein of 140 kDa . Spectrofluorimetric measurements in Fura - 2 loaded cells showed reduced CCE and Mn2 + entry in response to either thapsigargin or DB00136 SUB upon transfection with anti - Q13507 REA / 6/7 antisense oligodeoxynucleotides ( ODNs ) . Transfection with anti - P11473 REA antisense ODNs diminished DB00136 SUB - dependent Ca2 + and Mn2 + influx . Co-immunoprecipitation of Q13507 REA - like protein and P11473 REA under non-denaturating conditions was observed . We propose that endogenous Q13507 REA - like proteins and the P11473 REA participate in the modulation of CCE by DB00136 SUB in muscle cells , which could be mediated by an interaction between these proteins .

Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates . In this study , it is shown that short-term exposure of normal human marrow P28906 REA ( + ) P28907 REA ( - ) cells to low concentrations of tumor necrosis factor ( P01375 REA ) in the presence of 100 ng / mL P49771 REA and Steel factor and 20 ng / mL interleukin - 3 ( P08700 REA ) , P05231 REA , and granulocyte colony-stimulating factor , in either bulk or single-cell serum-free cultures , markedly reduces their ability subsequently to generate colony-forming cells ( CFCs ) in 6 - week stromal cell-containing long-term cultures without affecting their viability , mitogenic response , or short-term ability to produce CFCs . A similar differential effect on the functional attributes of P28906 REA ( + ) P28907 REA ( - ) cells was seen when P06681 REA - or P13671 REA - ceramide , but not dihydro - P06681 REA - ceramide ( an inactive analog of ceramide ) , was substituted for P01375 REA . The addition of D-erythro-MAPP ( a specific inhibitor of intracellular ceramide degradation ) enhanced the ability of P01375 REA to selectively eliminate long-term culture-initiating cell ( LTC-IC ) activity . These findings indicate that P01375 REA can directly modulate the ability of P28906 REA ( + ) P28907 REA ( - ) cells to maintain their LTC-IC function at doses below those required to initiate apoptosis , cell cycle arrest , or both , and they suggest that this may be mediated by the P01375 REA - induced generation of intracellular ceramide . Identification of a signaling intermediate that can influence primitive hematopoietic cell fate decisions offers a new approach to the investigation of signaling mechanisms in normal stem cell populations and to how these may be altered in leukemic cells .

Molecular evolution of the oxytocin-oxytocin receptor system in eutherians . DB00107 ( P01178 REA ) is a nine-amino-acid peptide hormone that is mainly released at the times of uterine contractions during parturition and milk ejection during lactation , whereas a similar peptide hormone , arginine vasopressin , primarily exerts direct antidiuretic action on the kidney and causes vasoconstriction of the peripheral vessels . The genes coding for these peptides are tandemly located on the same chromosome . A tandem duplication occurring in the common ancestor of jawed vertebrates has been proposed as responsible . In contrast to the two peptide hormones , only one oxytocin receptor ( P30559 REA ) but three arginine vasopressin receptors ( P37288 REA , P47901 REA , and P30518 REA ) are known ; these receptors probably arose from two rounds of genome duplication in the common ancestor of vertebrates . In this study , we addressed the molecular evolution of the P01178 REA - P30559 REA system in eutherians . Our analyses suggest that an amino acid change from isoleucine to lysine on the eighth site ( I8L ) of the peptide , which corresponded to a change from mesotocin to P01178 REA , had occurred during the common ancestral lineage of eutherians . At around the same time that the emergence of P01178 REA occurred , functional constraints on the P01178 REA receptor ( pre - P30559 REA ) might have relaxed , and a series of nonsynonymous substitutions might have accumulated . Only a few of these nonsynonymous substitutions might have contributed to reestablishing the molecular relationship between the P01178 REA ligand and its receptor , after which functional constraints on the P30559 REA were reinstated . Since the P01178 REA - P30559 REA system plays an important role in eutherians , the evolution of the P01178 REA - P30559 REA system was probably an essential component of the genesis of the eutherian signature .

DB06212 MEN , a selective oral vasopressin V2 receptor antagonist , ameliorates podocyte injury in puromycin aminonucleoside nephrotic rats . BACKGROUND : Proteinuria caused by glomerular disease is characterized by podocyte injury . P30518 REA antagonists are effective in reducing albuminuria , although their actions on glomerular podocytes have not been explored . The objective of this study was to evaluate the effects of tolvaptan , a selective oral V2 receptor antagonist , on podocytes in a puromycin aminonucleoside ( PAN ) - induced nephrosis rat model . METHODS : Rats were allocated to a control , PAN nephrosis , or tolvaptan-treated PAN nephrosis group ( n = 9 per group ) . Urinary protein excretion and serum levels of total protein , albumin , creatinine , and total cholesterol were measured on day 10 . The influence of tolvaptan on podocytes was examined in renal tissues by immunofluorescence and electron microscopy . RESULTS : PAN induced massive proteinuria and serum creatinine elevation on day 10 , both of which were significantly ameliorated by tolvaptan . Immunofluorescence studies of the podocyte-associated proteins nephrin and podocin revealed granular staining patterns in PAN nephrosis rats . In tolvaptan-treated rats , nephrin and podocin expressions retained their normal linear pattern . Electron microscopy showed foot process effacement was ameliorated in tolvaptan-treated rats . CONCLUSIONS : DB06212 MEN is protective against podocyte damage and proteinuria in PAN nephrosis . This study indicates that tolvaptan exerts a renoprotective effect by affecting podocyte morphology and probably function in PAN nephrosis . DB06212 MEN is a promising pharmacological tool in the treatment of renal edema .

The anti-proliferative effects of DB00136 SUB on breast and prostate cancer cells are associated with induction of P38398 REA gene expression . The anti-proliferative action of the seco-steroid hormone 1alpha , 25 - dihydroxyvitamin D3 [ DB00136 SUB ] extends to some , but not all breast and prostate cancer cell lines . By elucidating the molecular mechanisms mediating the sensitivity of these cells , we can identify critical target genes regulated directly or indirectly by DB00136 SUB and pathways potentially disrupted during transformation . In this study , we demonstrated the induction of expression of P38398 REA mRNA and protein as well as transcriptional activation from the P38398 REA - promoter by DB00136 SUB in the sensitive breast cancer cell line MCF - 7 . This was not observed in the DB00136 SUB - resistant breast cancer cell line MDA-MB - 436 . The induction of P38398 REA mRNA was blocked by cyclohexamide . This indicated that transcriptional activation was mediated indirectly by the vitamin D receptor ( P11473 REA ) . Inhibition of P11473 REA protein levels by stable transformation of the anti-sense P11473 REA in MCF - 7 reduced the sensitivity of MCF - 7 to DB00136 SUB by 50 - fold . In addition , the induction of P38398 REA protein and transcriptional activation of a P38398 REA promoter-luciferase reporter construct was abrogated in the stable transformant with the greatest reduction of P11473 REA levels . Examination of other breast and prostate cancer cell lines revealed that sensitivity to the anti-proliferative effects of 1alpha , 25 ( OH ) 2D3 was strongly associated with an ability to modulate P38398 REA protein . Furthermore , the expression of the estrogen receptor in these cell lines strongly correlated with their sensitivity to DB00136 SUB and their ability to modulate P38398 REA expression . Taken together , our data support a model whereby the anti-proliferative effects of DB00136 SUB are mediated , in part , by the induction of P38398 REA gene expression via transcriptional activation by factors induced by the P11473 REA and that this pathway is disrupted during the development of prostate and breast cancers .

Intracellular signaling pathways involved in the relaxin-induced proliferation of rat Sertoli cells . Regulation of Sertoli cell number is a key event to determine normal spermatogenesis . We have previously shown that relaxin and its G-protein coupled receptor Q9HBX9 are expressed in rat Sertoli cells , and that relaxin stimulates Sertoli cell proliferation . This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats . Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen ( P12004 REA ) , but did not affect the mRNA level of the differentiation markers cadherins 1 and 2 . Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK / P27361 REA / 2 or PI3K / AKT pathways , but not by inhibition of PKC or P00533 REA activity . Relaxin induced a rapid and transient activation of P27361 REA / 2 phosphorylation , which was MEK and P12931 REA - dependent , and involved upstream activation of G ( i ) . AKT activation could be detected 5 min after relaxin stimulation , and was still detected after 24h of stimulation with relaxin . Relaxin-induced AKT phosphorylation was G ( i ) - but not PKA-dependent , and it was blocked by both PI3K and MEK inhibitors . In conclusion , the mitogenic effect of relaxin in Sertoli cell involves coupling to G ( i ) and activation of both MEK / P27361 REA / 2 and PI3K / AKT pathways .

A new cell culture-based assay quantifies vitamin K 2,3- epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven Q9BQB6 assay . BACKGROUND : DB00682 MEN directly inhibits the vitamin K 2,3- epoxide reductase complex subunit 1 ( Q9BQB6 ) enzyme to effect anticoagulation . Q9BQB6 function has historically been assessed in vitro using a dithiothreitol ( DTT ) - driven vitamin K 2,3- epoxide reductase ( Q9BQB6 ) assay . DB00682 MEN inhibits wild-type Q9BQB6 function by the DTT - Q9BQB6 assay . However , Q9BQB6 variants with warfarin resistance-associated missense mutations often show low Q9BQB6 activities and warfarin sensitivity instead of resistance . OBJECTIVES : A cell culture-based , indirect Q9BQB6 assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant Q9BQB6 proteins . METHODS : Human coagulation factor ( F ) IX and Q9BQB6 variants were coexpressed in P29320 REA 293T cells under standardized conditions at various warfarin concentrations . Secreted FIX activity served as surrogate marker to report wild-type and variant Q9BQB6 inhibition by warfarin . RESULTS AND CONCLUSIONS : DB00682 MEN dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC 1 wild-type , Val 29Leu , Val 45Ala and Leu 128Arg variants . The corresponding calculated IC50 values were 24.7 , 136.4 , 152.0 and 1226.4 nm , respectively . Basal activities in the absence of warfarin for all Q9BQB6 variants were similar to that of wild-type Q9BQB6 . Ranked IC50 values from the cell culture-based assay accurately reflect elevated warfarin dosages for patients with Q9BQB6 missense mutation-associated warfarin resistance .

An essential role of the CAAT / enhancer binding protein-alpha in the vitamin D-induced expression of the human steroid / bile acid-sulfotransferase ( Q06520 REA ) . The vitamin D receptor ( P11473 REA ) regulates steroid and drug metabolism by inducing the genes encoding phase I and phase II enzymes . Q06520 REA is a liver - and intestine-expressed sulfo-conjugating enzyme that converts the alcohol-OH of neutral steroids , bile acids , and drugs to water-soluble sulfated metabolites . DB00136 SUB [ 1,25- ( OH ) 2D3 ] induces Q06520 REA gene transcription after the recruitment of P11473 REA to the vitamin D-responsive chromatin region of Q06520 REA . A composite element in human Q06520 REA directs the 1,25- ( OH ) 2D3 - mediated induction of natural and heterologous promoters . This element combines a P11473 REA / retinoid X receptor-alpha-binding site [ vitamin D response element ( VDRE ) ] , which is an imperfect inverted repeat 2 of AGCTCA , and a CAAT / enhancer binding protein ( C / EBP ) - binding site located 9 bp downstream to VDRE . The binding sites were identified by EMSA , antibody supershift , and deoxyribonuclease I footprinting . C / EBP-alpha at the composite element plays an essential role in the P11473 REA regulation of Q06520 REA , because 1 ) induction was lost for promoters with inactivating mutations at the VDRE or C / EBP element ; 2 ) Q06520 REA induction by 1,25- ( OH ) 2D3 in C / EBP-alpha-deficient cells required the expression of cotransfected C / EBP-alpha ; and 3 ) C / EBP-beta did not substitute for C / EBP-alpha in this regulation . P11473 REA and C / EBP-alpha were recruited concurrently to the composite element along with the coactivators p300 , steroid receptor coactivator 1 ( Q15788 REA ) , and P12931 REA - 2 , but not Q9Y6Q9 REA . P11473 REA and C / EBP-alpha associated endogenously as a DNA-dependent , coimmunoprecipitable complex , which was detected at a markedly higher level in 1,25- ( OH ) 2D3 - treated cells . These results provide the first example of the essential role of the interaction in cis between C / EBP-alpha and P11473 REA in directing 1,25- ( OH ) 2D3 - induced expression of a P11473 REA target gene .

P08571 REA receptor polymorphism and Alzheimer ' s disease risk . Activation of microglial cells is involved in the inflammatory component of Alzheimer ' s disease ( AD ) , and it may be triggered by infectious pathogens . P08571 REA , a receptor upregulated in activated microglia , plays a central role in innate immunity through recognition of bacterial lipopolysaccharide and initiation of inflammatory response . A polymorphism in the promoter region ( - 260 ) of the P08571 REA receptor has been found to be related to increased risk of bacterial infections and inflammatory diseases such as atherosclerosis . In a case-control study utilizing a clinically well-defined group of 310 sporadic AD patients and 310 control subjects , we investigated whether the P08571 REA ( - 260 ) polymorphism might be responsible for susceptibility to AD , and we also examined the combined gene effects between P08571 REA and P02649 REA and several other proinflammatory cytokine genes . The current study does not demonstrate an association between P08571 REA ( - 260 ) polymorphism and AD , neither through an independent effect nor through interaction with P02649 REA epsilon 4 allele or interleukin ( IL ) - 1A , P05231 REA , P10145 REA , tumor necrosis factor ( P01375 REA ) - alpha , and intercellular adhesion molecule - 1 polymorphisms .

Epigenetic marks define the lineage and differentiation potential of two distinct neural crest-derived intermediate odontogenic progenitor populations . Epigenetic mechanisms , such as histone modifications , play an active role in the differentiation and lineage commitment of DB05914 . In the present study , epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared : dental pulp ( DP ) and dental follicle ( DF ) . ChIP on chip assays revealed substantial H3K27me3 - mediated repression of odontoblast lineage genes Q9NZW4 and dentin matrix protein 1 ( DMP 1 ) in DF cells , but not in DP cells . Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells . In contrast , mineralization induction resulted in a highly dynamic histone modification response in DF cells , while there was only a subdued effect in DP cells . Both DF and DP progenitors featured H3K4me3 - active marks on the promoters of early mineralization genes Q13950 REA , P35548 REA , and P56178 REA , while Q8TDD2 , P21815 REA , and P02818 REA promoters were enriched for H3K9me3 or H3K27me3 . Compared to DF cells , DP cells expressed higher levels of three pluripotency-associated genes , Q01860 REA , Q9H9S0 REA , and P48431 REA . Finally , gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell-cell attachment genes in DP cells and neurogenesis genes in DF cells . In conclusion , the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of Q9NZW4 and DMP 1 genes and through dynamic histone enrichment responses to mineralization induction . Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages .

Stimulation of the peroxisome proliferator-activated receptor gamma ( Q07869 REA gamma ) and the expression of selected blood monocyte cytokine genes in diabetic macroangiopathy . Monocytes and macrophages play a key role in the progression of atheromatous changes . The peroxisome proliferator-activated receptor gamma ( Q07869 REA gamma ) can limit macroangiopathy through the control of cytokine transcription . The objectives of this study were to examine the influence of Q07869 REA gamma and its agonist ( rosiglitazone ) on the TNFalpha , P05231 REA , P10145 REA and P22301 REA gene expression in monocytes of patients with diabetic macroangiopathy and to analyse obtained results in context of selected atherogenic factors ant direct indicators of endothelial lesion . TNFalpha , P05231 REA , P10145 REA , P22301 REA and Q07869 REA gamma gene expression was assessed in peripheral blood monocytes in 45 patients with type 2 diabetes before and following 22 weeks of rosiglitazone therapy ( real-time PCR [ Applied Biosystems ] ) . As indicators of endothelial lesion , concentration of thrombomodulin ( immunoassay [ Diagnostica Stago ] ) and amount of circulating blood endothelial cells ( immunofluorescence method with MoAb Q8N0X4 - HEC 19 ) were determined . Following rosiglitazone therapy , a statistically significant downward tendency of TNFalpha ( p= 0.026 ) and P10145 REA ( p= 0.008 ) gene expression was noted . Before and following rosiglitazone treatment , Q07869 REA gamma , P05231 REA and P22301 REA gene expression was undetectable in studied monocytes in vivo . In conclusion , TNFalpha and P10145 REA play an important role in monocyte atherogenic activity . Rosiglitazone reduces monocyte proinflammatory readiness by influencing the expression of selected atherogenic cytokines ( Q07869 REA gamma-independent pathway ) .

Therapeutic efficacy of anti-ErbB 2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis . Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell . To generate single chain Fv ( scFv ) antibodies capable of triggering receptor-mediated endocytosis , we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell . Using this methodology , we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library . For this work , an immunotherapeutic was generated from one of these scFv ( P12259 REA ) , which bound to ErbB 2 ( P04626 REA / neu ) . The P12259 REA scFv was reengineered with a C-terminal cysteine , expressed at high levels in Escherichia coli , and coupled to sterically stabilized liposomes . P12259 REA anti-ErbB 2 immunoliposomes were immunoreactive as determined by surface plasmon resonance ( SPR ) and were avidly internalized by ErbB 2 - expressing tumor cell lines in proportion to the levels of ErbB 2 expression . P12259 REA - scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin . This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles .

Molecular targeting of the oncoprotein P53350 REA in pediatric acute myeloid leukemia : RO3280 , a novel P53350 REA inhibitor , induces apoptosis in leukemia cells . P53350 REA ( P53350 REA ) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs . RO3280 was recently identified as a novel P53350 REA inhibitor ; however its therapeutic effects in leukemia treatment are still unknown . We found that the P53350 REA protein was highly expressed in leukemia cell lines as well as 73.3 % ( 11/15 ) of pediatric acute myeloid leukemia ( AML ) samples . P53350 REA mRNA expression was significantly higher in AML samples compared with control samples ( 82.95 ± 110.28 vs . 6.36 ± 6.35 ; p < 0.001 ) . Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor P53350 REA expression ( p = 0.002 ) . The 50 % inhibitory concentration ( IC50 ) of RO3280 for acute leukemia cells was between 74 and 797 nM . The IC50 of RO3280 in primary acute lymphocytic leukemia ( ALL ) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM , respectively . RO3280 induced apoptosis and cell cycle disorder in leukemia cells . RO3280 treatment regulated several apoptosis-associated genes . The regulation of P43146 REA , P38936 REA , Q06187 REA , and O14508 REA was verified by western blot . These results provide insights into the potential use of RO3280 for AML therapy ; however , the underlying mechanisms remain to be determined .

Regulation of the human P38936 REA ( waf 1 / cip 1 ) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor . The main regulator of the human tumor suppresser gene P38936 REA ( waf 1 / cip 1 ) is the transcription factor p53 , but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor P11473 REA in the presence of its ligand 1alpha , 25 - dihydroxyvitamin D3 ( DB00136 SUB ) . To identify P11473 REA responding regions , we analyzed 20 overlapping regions covering the first 7.1 kb of the P38936 REA ( waf 1 / cip 1 ) promoter in MCF - 7 human breast cancer cells using chromatin immuno-precipitation assays ( ChIP ) with antibodies against p53 and P11473 REA . We confirmed two known p53 binding regions at approximate positions - 1400 and - 2300 and identified a novel site at position - 4500 . In addition , we found three P11473 REA - associated promoter regions at positions - 2300 , - 4500 and - 6900 , i . e . two regions showed binding for both p53 and P11473 REA . In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53 - positive promoter regions and also five DB00136 SUB response elements within the three P11473 REA - positive regions . Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5 - fluorouracil and DB00136 SUB . Moreover , re-ChIP assays confirmed the functionality of the three DB00136 SUB - reponsive promoter regions by monitoring simultaneous occupancy of P11473 REA with the co-activator proteins CBP , Q15788 REA and Q15648 REA . Taken together , we demonstrated that the human P38936 REA ( ( waf 1 / cip 1 ) ) gene is a primary DB00136 SUB - responding gene with at least three P11473 REA binding promoter regions , in two of which also p53 co-localizes .

Inhibition of histamine H1 receptor activity modulates proinflammatory cytokine production of dendritic cells through c-Rel activity . BACKGROUND : DB11320 exerts diverse effects on immune regulation through four types of histamine receptors ( HRs ) . Among them , type 1 receptor ( P35367 REA ) plays an important role in allergic inflammation . Dendritic cells ( DCs ) , which express at least three types of HRs , are professional antigen-presenting cells controlling the development of allergic inflammation . However , the molecular mechanisms involved in P35367 REA - mediated NF-ĸB signaling of DCs remain poorly defined . METHODS : Bone-marrow ( BM ) - derived DCs ( BM-DCs ) were treated with P35367 REA inverse agonists to interrupt basal P35367 REA - mediated signaling . The crosstalk of P35367 REA - mediated signaling and the NF-ĸB pathway was examined by NF-ĸB cellular activity using a luciferase reporter assay , NF-ĸB subunit analysis using Western blotting and P01375 REA - α promoter activity using chromatin immunoprecipitation . RESULTS : Blockage of P35367 REA signaling by inverse agonists significantly inhibited P01375 REA - α and P05231 REA production of BM-DCs . P35367 REA - specific agonists were able to enhance P01375 REA - α production , but this overexpression was significantly inhibited by NF-ĸB inhibitor . The P35367 REA inverse agonist ketotifen also suppressed cellular NF-ĸB activity , suggesting crosstalk between P35367 REA and NF-ĸB signaling in DCs . After comprehensive analysis of NF-ĸB subunits , c-Rel protein expression was significantly down-regulated in ketotifen-treated BM-DCs , which led to inhibition of the promoter activity of P01375 REA - α . Finally , adoptive transfer of the ketotifen-treated BM-DCs did not induce significant allergic airway inflammation compared to that of control cells in vivo . CONCLUSIONS : Our results suggest that c-Rel controls P35367 REA - mediated proinflammatory cytokine production in DCs . This study provides a potential mechanism of P35367 REA - mediated signaling and NF-ĸB pathway crosstalk in allergic inflammation .

Growth factor receptor / steroid receptor cross talk in trastuzumab-treated breast cancer . Treatment with tyrosine kinase inhibitors ( TKIs ) including trastuzumab has revolutionized the management of P04626 REA - positive breast cancer . Recent evaluation of clinical trial data suggests that a subset of P04626 REA / ER double-positive cancers may not receive significant benefit from the TKI therapy . Here we investigate the cross talk between P04626 REA and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc . In P04626 REA - positive breast cancer patients treated with neoadjuvant trastuzumab , steroid receptor-negative status ( ER and PR negative ) of pre-treatment biopsies predicted pathological complete response ( pCR ) ( n = 31 patients , P= 0.0486 ) , whereas elevated Myc protein inversely associated with pCR ( P= 0.0446 ) . Liquid chromatography mass spectrometry identified the corepressor Q9Y618 REA as a novel Myc-interacting protein . DB00072 MEN treatment enhanced Myc - Q9Y618 REA interactions in P04626 REA - overexpressing breast cancer cells ( LCC 1 ) and inhibited expression of the Myc target gene survivin . In P04626 REA - low , ER-positive steroid-dominant cells ( MCF 7 ) , trastuzumab therapy repressed Myc - Q9Y618 REA interactions and upregulated survivin expression . DB00072 MEN treatment induced ER-CBP interactions , enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2 . The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients ( n = 25 , P= 0.0089 ) and pS2 expression associated with residual cancer burden ( P= 0.0196 ) . Furthermore , metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive . In P04626 REA - overexpressing cells , trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable . However , with co-expression of the steroid pathway , this inhibition is lost and response to treatment is often poor .

[ Genetic basis for skeletal disease . Hereditary rickets ] . Hereditary rickets is caused by inborn error of vitamin D activation , vitamin D receptor ( P11473 REA ) function or increased urinary phosphate excretion . Loss-of-function mutation of 1alpha - hydroxylase gene and loss-of-function mutation of P11473 REA gene result in vitamin D-dependent rickets type I and type II , respectively . X-linked hypophosphatemic rickets ( XLH ) is the most common type of hypophosphatemic rickets , and autosomal dominant ( P30518 REA ) and negative ( ARHR ) types are rare . The diagnosis may be sometimes difficult and increasing cases of vitamin D deficiency must be distinguished .

Synthesis and biological activities of 14 - epi-MART - 10 and 14 - epi-MART - 11 : implications for cancer and osteoporosis treatment . The 14 - epimer of MART - 10 , namely 14 - epi-MART - 10 ( 14 - epi - 2alpha - ( 3 - hydroxypropyl ) - 1alpha , 25 - dihydroxy - 19 - norvitamin D3 ) and its 2 - epimeric analog ( 14 - epi-MART - 11 ) were efficiently synthesized using the Julia coupling reaction to connect between the P01031 REA and P13671 REA positions ( steroid numbering ) . An A-ring precursor was prepared from ( - ) - quinic acid as shown in the previous MART - 10 synthesis . The novel 14 - epi-CD-ring coupling partner with an elongated two carbon unit as a sulfone was synthesized from 14 - epi - 25 - hydroxy Grundmann ' s ketone in good yield . The subsequent coupling reaction followed by a deprotection step afforded a mixture of 14 - epi-MART - 10 and 14 - epi-MART - 11 in 40 % yield . To separate 14 - epi-MART - 10 and 14 - epi-MART - 11 , each primary hydroxyl group was esterified with a pivaloyl group and the resulting pivalates 2alpha and 2beta were separated by high performance liquid chromatography . After the separation , the P06681 REA - stereochemistry of each ( 2alpha or 2beta ) was determined by 1H NMR ( nuclear magnetic resonance ) studies including NOE ( nuclear Overhauser effect ) experiments . The pivaloyl group was removed under basic conditions to obtain the target molecules of 14 - epi-MART - 10 and 14 - epi-MART - 11 , respectively . The P11473 REA ( vitamin D receptor ) - binding affinity , HL - 60 ( human promyelocytic leukemia ) cell differentiation activity , antiproliferative activity in PZ-HPV - 7 ( immortalized normal prostate ) cells and transactivation activity of the osteocalcin promoter in Q9UKB1 ( human osteoblast cell line ) cells ( serum-free conditions ) were investigated . In addition , the effects on bone mineral density ( BMD ) and the blood and urine calcium concentrations of ovariectomized ( OVX ) rats were examined . 14 - epi-MART - 10 has much greater antiproliferative and cell differentiation activities compared to 1alpha , 25 - dihydroxyvitamin D3 ( DB00136 SUB ) .

Inhibitors of Q06187 REA and Q08881 REA : state of the new drugs for cancer , autoimmunity and inflammatory diseases . Q06187 REA and Q08881 REA are cytoplasmic tyrosine kinases of crucial importance for B and T cell development , with loss-of-function mutations causing X-linked agammaglobulinemia and susceptibility to severe , frequently lethal , Epstein-Barr virus infection , respectively . Over the last few years , considerable efforts have been made in order to develop small-molecule inhibitors for these kinases to treat lymphocyte malignancies , autoimmunity or allergy / hypersensitivity . The rationale is that even if complete lack of Q06187 REA or Q08881 REA during development causes severe immunodeficiency , inactivation after birth may result in a less severe phenotype . Moreover , therapy can be transient or only partially block the activity of Q06187 REA or Q08881 REA . Furthermore , a drug-induced B cell deficiency is treatable by gamma globulin substitution therapy . The newly developed Q06187 REA inhibitor P05154 REA - 32765 , recently renamed DB09053 MEN , has already entered several clinical trials for various forms of non-Hodgkin lymphoma as well as for multiple myeloma . Experimental animal studies have demonstrated highly promising treatment effects also in autoimmunity . Q08881 REA inhibitors are still under the early developmental phase , but it can be expected that such drugs will also become very useful . In this study , we present Q06187 REA and Q08881 REA with their signalling pathways and review the development of the corresponding inhibitors .

P15692 REA activates receptor-operated cation channels in human microvascular endothelial cells . OBJECTIVE : Vascular endothelial growth factor ( P15692 REA ) exerts many of its effects by stimulating endothelial calcium influx , but little is known about channels mediating P15692 REA - induced cation entry . The aim of this study was to measure and characterize for the first time the P15692 REA - activated cation current in human microvascular endothelial cells ( HMVECs ) . METHODS AND RESULTS : Whole-cell patch-clamp recordings were made from HMVECs . During applied voltage ramps , P15692 REA activated a current that reversed at 0 mV , was sensitive to gadolinium , and required extracellular cations . Noise analysis yielded a single-channel conductance of 27 pS . The current was not dependent on intracellular calcium stores , and was not blocked by inositol triphosphate ( IP3 ) receptor or serine / threonine kinase inhibition but was partially inhibited by flufenamic acid . A similar current was activated by 1 - oleoyl - 2 - acetyl-sn-glycerol ( OAG ) , a membrane-permeant analog of diacylglycerol ( DAG ) . To determine whether P15692 REA could activate recombinant ion channels with similar properties , we investigated the effect of P15692 REA on Chinese hamster ovary cells cotransfected with P35968 REA and the canonical transient receptor potential ( TRPC ) channels , Q13507 REA or Q9Y210 REA . P15692 REA induced a similar current to that described above in P35968 REA - Q13507 REA and P35968 REA - Q9Y210 REA cells but not in cells transfected with either cDNA alone . CONCLUSIONS : P15692 REA activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells . P15692 REA is also able to activate heterologously expressed Q13507 REA / 6 channels through P35968 REA .

Orai proteins interact with TRPC channels and confer responsiveness to store depletion . The TRPC ( C-type transient receptor potential ) class of ion channels has been hypothesized to participate in store-operated Ca ( 2 + ) entry ( SOCE ) . Recently , however , Q13586 REA and Orai 1 proteins have been proposed to form SOCE channels . Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored . Here we show that Orai 1 physically interacts with the N and C termini of Q13507 REA and Q9Y210 REA , and that in cells overexpressing either Q13507 REA or Q9Y210 REA in a store-depletion insensitive manner , these TRPCs become sensitive to store depletion upon expression of an exogenous Orai . Thus , Orai - 1 , - 2 , and - 3 enhanced thapsigargin-induced calcium entry by 50-150 % in cells stably overexpressing either Q13507 REA or Q9Y210 REA . Orai 1 expression had no significant effect on endogenous , thapsigargin-induced calcium entry in wild-type cells ( P29320 REA - 293 , COS 1 ) , in P29320 REA cells expressing a thapsigargin-sensitive variant of Q13507 REA ( TRPC 3a ) , or in P29320 REA cells overexpressing another membrane protein , P37288 REA . Single-channel cation currents present in membrane patches of Q13507 REA - overexpressing cells were suppressed by expression of Orai 1 . We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer Q13586 REA - mediated store depletion sensitivity to these channels .