interacts_with DB00091?
Characterization of plant P18887 and its interaction with proliferating cell nuclear antigen . In plants , there are no P06746 ( Pol beta ) and P49916 ( Lig3 ) genes . Thus , the plant short-patch base excision repair ( short-patch BER ) pathway must differ considerably from that in mammals . We characterized the rice ( Oryza Sativa L. cv. Nipponbare ) homologue of the mammalian X-ray repair cross complementing 1 ( P18887 ) , a well-known BER protein . The plant P18887 lacks the N-terminal domain ( NTD ) which is required for Pol beta binding and is essential for mammalian cell survival . The recombinant rice P18887 ( OsXRCC1 ) protein binds single-stranded DNA ( ssDNA ) as well as double-stranded DNA ( dsDNA ) and also interacts with rice proliferating cell nuclear antigen ( OsPCNA ) in a pull-down assay . Through immunoprecipitation , we demonstrated that OsXRCC1 forms a complex with P12004 in vivo . OsXRCC1 mRNA was expressed in all rice organs and was induced by application of bleomycin , but not of MMS , H(2)O(2) or UV-B . DB00290 also increased the fraction of OsXRCC1 associated with chromatin . These results suggest that OsXRCC1 contributes to DNA repair pathways that differ from the mammalian BER system . P62937 is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro . DB00091 ( DB00091 ) and its derivatives potently suppress hepatitis C virus ( HCV ) replication . Recently , DB00091 -resistant HCV replicons have been identified in vitro . We examined the dependence of the wild-type and DB00091 -resistant replicons on various cyclophilins for replication . A strong correlation between DB00091 resistance and reduced dependency on cyclophilin A ( CyPA ) for replication was identified . Silencing of CyPB or CyPC expression had no significant effect on replication , whereas various forms of small interfering RNA ( siRNA ) directed at CyPA inhibited HCV replication of wild-type but not DB00091 -resistant replicons . The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication , and expression of an siRNA-resistant CyPA cDNA rescued replication . In addition , an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay . Depletion of CyPA alone in the DB00091 -resistant replicon cells eliminated DB00091 resistance , indicating that CyPA is the chief mediator of the observed DB00091 resistance . The dependency on CyPA for replication was observed for both genotype ( GT ) 1a and 1b replicons as well as a GT 2a infectious virus . An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to DB00091 treatment in wild-type but not in resistant replicons was detected . These findings reveal the molecular mechanism of DB00091 resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection . DB06287 induces surfactant lipid accumulation and lung inflammation in mice . Interstitial lung disease ( ILD ) is a well-known adverse effect of mammalian target of rapamycin ( P42345 ) inhibitors . However , it remains unknown how lung toxicities are induced by P42345 inhibitors . Here , we constructed a mouse model of P42345 inhibitor-induced ILD using temsirolimus and examined the pathogenesis of the disease . Male ICR mice were treated with an intraperitoneal injection of different doses of temsirolimus ( 3 or 30 mg·kg(-1)·wk(-1) ) or vehicle . DB06287 treatment increased capillary-alveolar permeability and induced neutrophil infiltration and fibrinous exudate into the alveolar space , indicating alveolar epithelial and/or endothelial injury . It also induced macrophage depletion and the accumulation of excessive surfactant phospholipids and cholesterols . Alveolar macrophage depletion is thought to cause surfactant lipid accumulation . To further examine whether temsirolimus has cytotoxic and/or cytostatic effects on alveolar macrophages and alveolar epithelial cells , we performed in vitro experiments . DB06287 inhibited cell proliferation and viability in both alveolar macrophage and alveolar epithelial cells . DB06287 treatment caused some signs of pulmonary inflammation , including upregulated expression of several proinflammatory cytokines in both bronchoalveolar lavage cells and lung homogenates , and an increase in lymphocytes in the bronchoalveolar lavage fluid . These findings indicate that temsirolimus has the potential to induce alveolar epithelial injury and to deplete alveolar macrophages followed by surfactant lipid accumulation , resulting in pulmonary inflammation . This is the first study to focus on the pathogenesis of P42345 inhibitor-induced ILD using an animal model . Biological and immunological studies of bovine hypothalamic DB05394 . P06850 B ( CRF-B ) is a peptide(s) isolated from bovine hypothalamic extracts by Sephadex G-100 chromatography on the basis of its ability to stimulate secretion of adrenocorticotropin ( DB01285 ) in vitro and in vivo . It is similar in molecular size to the 41-residue ovine CRF ( oCRF ) or rat CRF ( rCRF ) recently elucidated and appears to be their bovine counterpart . Immunoreactivity of CRF-B was examined in homologous radioimmunoassays ( RIAs ) for oCRF or rCRF , using several anti-oCRF and anti-rCRF antibodies . CRF-B cross-reacted well with anti-oCRF antibodies but poorly with anti-rCRF antibodies . Purification of CRF-B with preparative isoelectric focusing yielded four CRF peaks , B-1 ( pH 4.7 ) , B-2 ( pH 5.5 ) , B-3 ( pH 6.3 ) , and B-4 ( pH 7.0 ) , which accounted for 16 , 30 , 46 , and 8 % of the total immunoreactivity , respectively . CRF B-2 , B-3 , and B-4 showed both immunological activity and biological activity in vitro ( cell culture assay ) and in vivo ( Arimura assay ) , whereas CRF B-1 showed only immunoreactivity . Their relative bioactivity/immunoreactivity ratios were 0 ( B-1 ) , 1 ( B-2 ) , 1 ( B-3 ) , and 3 ( B-4 ) . All of these CRF-B subtypes exhibited RIA displacement curves parallel to that for the oCRF standard and coeluted with oCRF on Sephadex G-100 chromatography , which suggests that their molecular modifications are relatively minor . P62937 -deficient mice are resistant to immunosuppression by cyclosporine . DB00091 is an immunosuppressive drug that is widely used to prevent organ transplant rejection . Known intracellular ligands for cyclosporine include the cyclophilins , a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells . Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin , a serine/threonine phosphatase that is activated by TCR engagement . Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified . Most of these residues are highly conserved within the 15 mammalian cyclophilins , suggesting that many are potential targets for the drug . We examined the effects of cyclosporine on immune cells and mice lacking Ppia , the gene encoding the prototypical cyclophilin protein cyclophilin A . TCR-induced proliferation and signal transduction by Ppia(-/-) P01730 (+) T cells were resistant to cyclosporine , an effect that was attributable to diminished calcineurin inhibition . Immunosuppressive doses of cyclosporine failed to block the responses of Ppia(-/-) mice to allogeneic challenge . Rag2(-/-) mice reconstituted with Ppia(-/-) splenocytes were also cyclosporine resistant , indicating that this property is intrinsic to Ppia(-/-) immune cells . Thus , among multiple potential ligands , CypA is the primary mediator of immunosuppression by cyclosporine . P62937 and calcineurin functions investigated by gene inactivation , cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea . Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence . Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation , drug inhibition and cDNA macroarrays approaches . First , the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination . The bcp1Delta null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves . Opposite to this , calcineurin inhibition using cyclosporin A ( DB00091 ) modified hyphal morphology and prevented infection structure formation . DB00091 drug pattern signature on macroarrays allowed the identification of 18 calcineurin-dependent ( CND ) genes among 2839 B. cinerea genes . Among the co-regulated CND genes , three were shown to be organized as a physical cluster that could be involved in secondary metabolism . The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin-dependent ( O75976 ) genes that were different from CND genes . Finally , no DB00091 drug pattern signature was observed in the bcp1Delta null mutant which provided a molecular target validation of the drug . Marine invertebrates cross phyla comparisons reveal highly conserved immune machinery . Naturally occurring histocompatibility responses , following tissue-to-tissue allogeneic contacts , are common among numerous colonial marine invertebrate taxa , including sponges , cnidarians , bryozoans and ascidians . These responses , often culminating in either tissue fusions or rejections , activate a wide array of innate immune components . By comparing two allorejection EST libraries , developed from alloincompatible challenged colonies of the stony coral Stylophora pistillata and the ascidian Botryllus schlosseri , we revealed a common basis for innate immunity in these two evolutionary distant species . Two prominent genes within this common basis were the immunophilins , P62937 ( CypA ) and FK506-binding protein ( FKBP ) . In situ hybridizations revealed that mRNA expression of the coral and ascidian immunophilins was restricted to specific allorecognition effector cell populations ( nematoblasts and nematocytes in the coral and morula cells in the ascidian ) . The expressions were limited to only some of the effector cells within a population , disclosing disparities in numbers and location between naïve colonies and their immune challenged counterparts . Administration of the immunosuppression drug DB00091 -A during ascidian 's allogeneic assays inhibited both fusion and rejection reactions , probably through the inhibition of ascidian 's immunocytes ( morula cells ) movement and activation . Our results , together with previous published data , depict an immunophilins-based immune mechanism , which is similarly activated in allogeneic responses of distantly related animals from sponges to humans . Genetic factors influencing outcome from neurotrauma . PURPOSE OF REVIEW : Clinical outcome after neurotrauma is considerably variable and can only partly be explained by known prognostic factors . There is converging evidence from genetic research that a number of genetic variants may contribute to this variability . This review provides recent data from human studies , published in the previous year , on genetic factors influencing outcome after neurotrauma . The bibliographic databases MEDLINE , EMBASE and PsycINFO were searched to identify relevant studies . RECENT FINDINGS : Genetic susceptibility to various aspects of clinical outcome after neurotrauma was reported in recent clinical studies . Genetic loci investigated include polymorphisms in P02649 , P21397 , P23560 , NOS3 , P05231 , P12036 , P31645 , P21964 , P48454 and Q8IX03 genes . The importance of these findings and future directions are discussed . SUMMARY : Recent genetic studies have revealed emerging aspects and extended the existing knowledge regarding the pathogenesis of neurotrauma and the genetic influence on phenotypic diversity . A better understanding of the underlying biological pathways and molecular mechanisms of an individual 's response to neurotrauma may hold the promise of novel treatment strategies and improved clinical outcome . SERCA2b activity is regulated by cyclophilins in human platelets . OBJECTIVE : The role of cyclophilins ( chaperones that are widely expressed in different cell types , including human platelets ) was explored in sarcoendoplasmic calcium ( Ca(2+) ) adenosine triphosphatase ( SERCA ) activity . METHODS AND RESULTS : Cyclophilin inhibition by cyclosporin A ( DB00091 ) evoked a time- and concentration-dependent reduction of Ca(2+) uptake by SERCA2b . However , other Ca(2+)-adenosine triphosphatases expressed in platelets , such as Q93084 and plasma membrane Ca(2+) adenosine triphophatase , remained unaltered after DB00091 treatment . Cypermethrin , a non- DB00091 -related calcineurin inhibitor , did not alter SERCA2b activity . Furthermore , SERCA2b was affected by other DB00091 analogues , which do not interfere with calcineurin , such as PKF-211-811-NX5 ( NIM811 ) and sanglifehrin A . Inhibition of the immunophilin family members using FK506 ( tacrolimus ) did not alter SERCA2b ability to sequester Ca(2+) into the dense tubular system . Coimmunoprecipitation experiments confirmed that cyclophilin A associates with SERCA2b and stromal interaction molecule-1 in resting platelets . This interaction is attenuated by the physiological agonist thrombin but enhanced by treatment with DB00091 or sanglifehrin A . CONCLUSIONS : P62937 is a regulator of SERCA2b in human platelets . Cyclophilin inhibitors as a novel HCV therapy . A critical role of Cyclophilins , mostly P62937 ( CyPA ) , in the replication of HCV is supported by a growing body of in vitro and in vivo evidence . CyPA probably interacts directly with nonstructural protein 5A to exert its effect , through its peptidyl-prolyl isomerase activity , on maintaining the proper structure and function of the HCV replicase . The major proline substrates are located in domain II of NS5A , centered around a " DY " dipeptide motif that regulates CyPA dependence and DB00091 resistance . Importantly , DB00091 A derivatives that lack immunosuppressive function efficiently block the CyPA-NS5A interaction and inhibit HCV in cell culture , an animal model , and human trials . Given the high genetic barrier to development of resistance and the distinctness of their mechanism from that of either the current standard of care or any specifically targeted antiviral therapy for HCV ( P35610 -C ) , CyP inhibitors hold promise as a novel class of anti-HCV therapy . DB00091 and sanglifehrin A enhance chemotherapeutic effect of cisplatin in P13671 glioma cells . Glioma is the most common type of brain tumors in adults , and treatment of high-grade gliomas is still palliative . Studies to date have revealed only modest effect in attenuating growth of these tumors with single agent therapy , but combination treatment appears to be more effective . P62937 ( CypA ) , a target of immunosuppressive drugs cyclosporin A ( DB00091 ) and sanglifehrin A ( SFA ) , is an intracellular protein that has peptidyl-prolyl cis-trans isomerase ( PPIase ) enzymatic activity . Previously , we showed that overexpressed CypA induced chemoresistance in cancer cells . Here we provide evidence that combination of cisplatin with either DB00091 or SFA synergistically enhances apoptotic cell death in P13671 glioma cells , compared with single agent treatment . Enhanced apoptotic cell death is a result of an increase in ROS generation and a decrease in intracellular glutathione levels . Consistently , CypA knockdown by siRNA also enhances cisplatin-induced apoptosis . Immunohistochemical analysis showed increased expression of CypA in human glioblastoma multiforme , but not in normal human astrocytes . CypA was also shown to be up-regulated in P13671 glioma cells during hypoxia . In conclusion , DB00091 or SFA in combination with cisplatin synergistically enhances cisplatin-induced apoptosis in P13671 glioma cells via inhibition of PPIase activity of CypA , indicating that development of new drugs that selectively inhibit the CypA PPIase activity without immune suppression may facilitate alleviation of chemoresistance in treatment of high-grade glioma . QTL alleles on chromosome 7 from fatty Meishan pigs reduce fat deposition . For detecting QTL in the whole swine genome , 1068 pigs from three F2 populations constructed by crossing European Wild boar and Pietrain ( W x P ) , Meishan and Pietrain ( M x P ) , and Wild Boar and Meishan ( W x M ) were genotyped for genetic markers evenly spaced at approximately 20 cM intervals . AQTL analysis was performed using a least-squares method . Here the results of the QTL analysis on the porcine chromosome 7 are presented . QTL for carcass composition ( e.g. head weight , carcass length , backfat depth , abdominal fat and bacon meat ) were mapped in the chromosomal region P62937 / Q08752 - P01374 -S0102 in M x P and W x M , but not in W x P . The QTL explained 5.3 % -27.2 % of the F2 phenotypic variance in the two F2 populations . Most traits affected by the mapped QTL were related to carcass fatness . The mode of gene action of QTL was additive . Surprisingly , in contrast to the parental phenotype , the QTL alleles from fatty Meishan were associated with thinner backfat than Pietrain and Wild Boar alleles , suggesting that the genome of the fatty Meishan pig contains genes which can reduce fat content of carcass substantially . Effect of valproic acid through regulation of DB01221 receptor- P29323 signaling in sleep deprivation rats . Although the effect of mood stabilizer valproic acid ( DB00313 ) through multiple signaling pathways has been shown , its therapeutic mechanism is still largely unknown . We investigated the effect of DB00313 ( 200 mg/kg , every 12 h ) in sleep deprivation ( SD ) rats ( 72 h ) , the manic-like animal model , focusing on the N-methyl-D : -aspartic acid ( DB01221 ) receptor and signaling mediators of synaptic plasticity such as extracellular signal-regulated protein kinase ( P29323 ) , DB02527 response element-binding protein ( CREB ) , B cell chronic lymphocytic leukemia/lymphoma 2 ( P10415 ) , and brain-derived neurotrophic factor ( P23560 ) . SD reduced the expression of the Q13224 subunit of the DB01221 receptor in the frontal cortex and hippocampus but did not affect the expression of Q9UHB4 and Q12879 subunits . In comparison , DB00313 inhibited the SD-induced reduction of Q13224 expression in both brain regions . In addition , SD attenuated P29323 phosphorylation in the frontal cortex and hippocampus , whereas DB00313 prevented the attenuation . DB00313 also protected the SD-induced decrease of CREB phosphorylation , P10415 expression , and P23560 expression in the frontal cortex but not in the hippocampus . These results indicate that DB00313 could regulate DB01221 receptor- P29323 signaling in SD rats , preventing the SD-induced decrease of the expression of Q13224 subunit and the activation of P29323 signaling mediators such as P29323 , CREB , P10415 , and P23560 . Inhibition of the lytic activity of perforin ( cytolysin ) and of late complement components by proteoglycans . The complement components ( P13671 , P10643 , Q99618 and P02748 ) implicated in the lysis of target cells and the pore-forming , lytic protein from cytotoxic T-lymphocytes and NK-cells , perforin , contain an amino acid sequence which is highly homologous to a repeat unit identified in the LDL-receptor ( Tschopp et al. , 1986 , Nature , 322 , 831-834 ) . The domain of the LDL-receptor , which is thought to interact with a positively charged segment of its ligands apoprotein B and E , is rich in cysteine residues and contains a cluster of negative charges . We show that the negatively charged molecules suramin and glycosaminoglycans , the positively charged peptides protamine and polylysine , all of which are known to abolish binding of LDL to its receptor ( Goldstein et al. , 1985 , A. Rev. cell. Biol. , 1 , 1-39 ) inhibit the lytic activities of P13671 , P10643 , Q99618 , P02748 and perforin . Moreover , these negatively charged molecules are potent inhibitors of cytolytic T-lymphocyte-mediated lysis of target cells , suggesting a functionally crucial role for perforin in cell-mediated cytolysis . We propose that the negatively charged , cysteine-rich domain of these complement proteins and perforin interacts with an as yet unidentified positively charged segment of its ligand in a manner analogous to the LDL- P01130 interaction . Homologous cysteine-rich domains in functionally unrelated proteins may therefore be functionally conserved as ideal rigid interaction domains with the conserved cysteine residues as framework . Specificity of the domain for its ligand would be conferred by the non-conserved amino acid residues . P62937 as a target of DB00515 chemosensitizers . Platinum-based chemotherapeutics are the mainstay of treatment of a range of tumors achieving high response rates but limited in the course of disease by appearance of drug resistance . Tumor cells respond with reduced uptake and increased intracellular inactivation of the drugs , as well as increased DNA repair and general resistance to chemotherapyinduced cell death . DB00515 is known to induce expression of cyclophilins , a group of proteins that have peptidyl-prolyl cis-trans isomerase ( PPIase ) and molecular chaperone activities , as stress response . P62937 ( CypA ) and other members of this family are inhibited by cyclosporin A ( DB00091 ) which sensitized diverse drug-resistant tumor cell lines in vitro to cisplatin . This effect of DB00091 was attributed to metabolic changes , inhibition of DNA repair , enhancement of apoptosis , altered intracellular signal transduction or increased production of reactive oxygen species ( ROS ) , although no definitive explanation was provided so far . Several clinical trials employing cisplatin/carboplatin in combination with DB00091 yielded unsatisfactory results . Since viral replication was found to be dependent on cyclophilins of the host cells , effective new inhibitors , different from DB00091 or with low or absent immunosuppressive activity , are in development or clinical trials . Sanglifehrins are more potent than DB00091 and proved to increase toxicity of cisplatin against hepatocellular cancer cells in vitro . These novel cyclophilin inhibitors may offer new opportunities to achieve reversal of resistance to platinumbased drugs in refractory patients . Responsive cancer patients may be enriched in clinical trials by an identification of the downstream targets of Cyps responsible for chemoresistance . Insights into the roles of cyclophilin A during influenza virus infection . P62937 ( CypA ) is the main member of the immunophilin superfamily that has peptidyl-prolyl cis-trans isomerase activity . CypA participates in protein folding , cell signaling , inflammation and tumorigenesis . Further , CypA plays critical roles in the replication of several viruses . Upon influenza virus infection , CypA inhibits viral replication by interacting with the M1 protein . In addition , CypA is incorporated into the influenza virus virions . Finally , DB00091 ( DB00091 ) , the main inhibitor of CypA , inhibits influenza virus replication through CypA-dependent and -independent pathways . This review briefly summarizes recent advances in understanding the roles of CypA during influenza virus infection . Anti-inflammatory effect of transduced PEP-1-cyclophilin A in Raw264.7 cells and 12-O-tetradecanoylphorbol-13-acetate-induced mice . AIMS : P62937 ( CypA ) is an immunophilin that acts as a receptor for the immunosuppressant drug cyclosporine A ( DB00091 ) . CypA has emerged as a potential drug target for several inflammatory diseases , although the details of its mechanism are unclear . We examined the protective effects of CypA on inflammation in Raw 264.7 cells and animal models . MAIN METHODS : A human CypA gene was fused with a protein transduction domain , PEP-1 peptide , to construct a cell permeable PEP-1-CypA protein . The protein expression level of cyclooxygenase-2 ( P35354 ) and cytokines was detected by Western blot , ELISA and mRNA level of P35354 and cytokines were measured by RT-PCR . The nuclear factor-kappa B ( NF-kB ) and mitogen-activated protein kinase ( MAPK ) activation were analyzed by Western blot and electrophoretic mobility shift assay . Skin inflammation was detected with immunohistochemistry . KEY FINDINGS : Transduced PEP-1-CypA protein markedly inhibited lipopolysaccharide- and 12-O-tetradecanoyl phorbol-13-acetate-induced expression levels of P35354 as well as pro-inflammatory cytokine levels in vitro and in vivo . Furthermore , transduced PEP-1-CypA protein resulted in a significant reduction in the activation of NF-kB and MAPK . SIGNIFICANCE : The results indicate that PEP-1-CypA inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK activation upon stimulation of inflammation in vitro and in vivo . PEP-1-CypA protein may potentially be used as a therapeutic agent against skin diseases-related inflammation . Behavioural and hypothalamic molecular effects of the anti-cancer agent cisplatin in the rat : A model of chemotherapy-related malaise ? Many cancer patients receiving chemotherapy experience fatigue , disturbed circadian rhythms , anorexia and a variety of dyspeptic symptoms including nausea . There is no animal model for this ' chemotherapy-related malaise ' so we investigated the behavioural and molecular effects of a potent chemotherapeutic agent , cisplatin ( CP , 6 mg/kg , i.p. ) in rats . Dark-phase horizontal locomotor activity declined post-CP reaching a nadir on day 3 ( P < 0.001 ) , before recovering after 7 days . CP 's effect was most marked in the late part ( 05.00-07.00 ) of the dark-phase . Food intake reached a nadir ( P > 0.001 ) at 2 days , coincident with an increase in gastric contents ( cisplatin 9.04+/-0.8 vs. saline 2.32+/-0.3 g ; P < 0.001 ) . No changes occurred in hypothalamic mRNA expression for O00253 , P01303 , O43612 , P06850 , IL-1 , P05231 , TNFalpha , P45844 , P31645 , P62937 and P00492 mRNA but tryptophan hydroxylase ( P17752 ) mRNA was decreased ( 47 % , P < 0.05 ) at day 21 post-CP . This shows that despite marked behavioural effects of cisplatin , only a discrete change ( P17752 ) was found in hypothalamic mRNA expression and that occurred when the animals ' behaviour had recovered . Findings are discussed in relation to the neuropharmacology of chemotherapy-induced malaise . Structure of the human Q9H244 receptor in complex with an antithrombotic drug . P2Y receptors ( P2YRs ) , a family of purinergic G-protein-coupled receptors ( GPCRs ) , are activated by extracellular nucleotides . There are a total of eight distinct functional P2YRs expressed in human , which are subdivided into P47900 -like receptors and Q9H244 -like receptors . Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo , which limits our understanding of this receptor family . P2Y12R regulates platelet activation and thrombus formation , and several antithrombotic drugs targeting P2Y12R -- including the prodrugs clopidogrel ( Plavix ) and prasugrel ( DB06209 ) that are metabolized and bind covalently , and the nucleoside analogue ticagrelor ( DB08816 ) that acts directly on the receptor -- have been approved for the prevention of stroke and myocardial infarction . However , limitations of these drugs ( for example , a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor ) suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors . Here we report the 2.6 Å resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist , AZD1283 . The structure reveals a distinct straight conformation of helix V , which sets P2Y12R apart from all other known class A GPCR structures . With AZD1283 bound , the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic . Along with the details of the AZD1283-binding site , analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding . The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates . Design and synthesis of conformationally constrained cyclophilin inhibitors showing a cyclosporin-A phenotype in C. elegans . P62937 ( CypA ) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A ( DB00091 ) . Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template . Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands . Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans . The use of the VerifyNow Q9H244 point-of-care device to monitor platelet function across a range of Q9H244 inhibition levels following prasugrel and clopidogrel administration . Variability in response to antiplatelet agents has prompted the development of point-of-care ( POC ) technology . In this study , we compared the VerifyNow Q9H244 ( VN- Q9H244 ) POC device with light transmission aggregometry ( P01374 ) in subjects switched directly from clopidogrel to prasugrel . Healthy subjects on aspirin were administered a clopidogrel 600 mg loading dose ( LD ) followed by a 75 mg/d maintenance dose ( MD ) for 10 days . Subjects were then switched to a prasugrel 60 mg LD and then 10 mg/d MD for 10 days ( n = 16 ) , or to a prasugrel 10 mg/d MD for 11 days ( n = 19 ) . Platelet function was measured by P01374 and VN- Q9H244 at baseline and after dosing . DB00758 600 mg LD/75 mg MD treatment led to a reduction in P2Y(12) reaction units ( PRU ) from baseline . A switch from clopidogrel MD to prasugrel 60 mg LD/10 mg MD produced an immediate decrease in PRU , while a switch to prasugrel 10 mg MD resulted in a more gradual decline . Consistent with the reduction in PRU , device-reported percent inhibition increased during both clopidogrel and prasugrel regimens . Inhibition of platelet aggregation as measured by P01374 showed a very similar pattern to that found with VN- Q9H244 measurement , irrespective of treatment regimens . The dynamic range of VN- Q9H244 appeared to be narrower than that of P01374 . With two different thienopyridines , the VN- Q9H244 device , within a somewhat more limited range , reflected the overall magnitude of change in aggregation response determined by P01374 . The determination of the clinical utility of such POC devices will require their use in clinical outcome studies . DB00091 regulates the levels of cyclophilin A in neuroblastoma cells in culture . P62937 ( CyP-A ) , a member of a highly conserved family of proteins , immunophilins , is the major intracellular receptor for the immunosuppressive drug , cyclosporin A ( DB00091 ) . CyP-A is widely expressed in many tissues , but is found in the highest concentration in brain tissues and may perform critical neuronal functions . DB00091 is a known neurotoxin . Therefore , understanding the regulation of CyP-A levels in nerve cells , particularly by DB00091 , is important . We have utilized murine neuroblastoma ( NB ) cells as an experimental model to investigate this issue . Our results show that DB00091 alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining . However , inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate ( DB02527 ) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone ( RO20-1724 ) , an inhibitor of cyclic nucleotide phosphodiesterase , or prostaglandin E1 ( PGE1 ) , a stimulator of adenylate cyclase , was not sufficient to increase CyP-A levels . DB00091 was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells . Increases in CyP-A levels , however , occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using ( CyP-A ) -specific primers . These results suggest that DB00091 regulates the level of its own binding protein , CyP-A , in both undifferentiated and DB02527 -induced differentiated NB cells in culture . Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity . DB00091 ( DB00091 ) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug . P62937 ( CypA ) is the most abundantly and ubiquitously expressed family member of cyclophilins . We previously showed that DB00091 toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase ( PPIase ) activity of CypA in DB00091 -treated myoblasts [ FASEB J. 16 ( 2002 ) 1633 ] . Since DB00091 -induced nephrotoxicity is the most significant adverse effect in its clinical utilization , we here investigated the role of DB00091 inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models . Transgenic mice of either wild type ( CypA/wt ) or R55A PPIase mutant type ( CypA/R55A ) , a dominant negative mutant of CypA PPIase activity , showed normal growth without any apparent abnormalities . However , DB00091 -induced nephrotoxicity was virtually suppressed in CypA/wt mice , but exacerbated in CypA/R55A mice , compared to that of littermates . Also , life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during DB00091 administration . Besides , DB00091 -induced nephrotoxicity was inversely related to the levels of catalase expression and activity . In conclusion , our data provide in vivo evidence that supplement of CypA PPIase activity allows animal 's resistance toward DB00091 -induced nephrotoxicity . Purinergic P47900 receptor signaling mediates wound stimuli-induced cyclooxygenase-2 expression in intestinal subepithelial myofibroblasts . Intestinal subepithelial myofibroblasts ( ISMFs ) are crucial for barrier formation against inflammatory stimuli . Physical injury induces cyclooxygenase-2 ( P35354 ) expression , which accelerates wound healing by ISMFs . However , the mechanism of P35354 induction remains unclear . Physically damaged cells release DB00171 . Here , we investigate the role of DB00171 -purinergic signaling in wound-induced P35354 induction in ISMFs . By 24h post-injury , bovine ISMFs had migrated to and closed the wounded area . A P36551 inhibitor , indomethacin or a purinergic P2 receptor antagonist , suramin , inhibited wound healing . However , additional treatment with indomethacin did not influence wound healing in suramin-treated ISMFs . RT-PCR showed an increase in P35354 mRNA expression 2h post-injury , which was inhibited by suramin . These results suggest that DB00171 mediates wound-induced P35354 elevation . We next assessed the contribution of various purinergic receptors in P35354 induction . An DB00171 analog , ATPγS and a purinergic P47900 , 11-13 receptors agonist , ADP , were among the agents tested which increased P35354 expression . ATPγS-induced P35354 mRNA expression was suppressed by suramin or a purinergic P2Xs , P47900 , 4 , 6 , and 13 receptors antagonist , PPADS . These data suggest the involvement of Gq-coupled purinergic P47900 receptor or Gi-coupled purinergic Q9BPV8 receptor in P35354 induction . U73122 , an inhibitor of phospholipase C , which is a downstream signal of Gq protein , showed suppression of P35354 mRNA expression . However , pertussis toxin , a Gi inhibitor , did not show suppression . We also revealed that inhibitors of p38 MAPK and PKC inhibited ATPγS-induced P35354 mRNA expression . Collectively , purinergic P47900 receptor signaling mediates wound-induced P35354 expression through p38 MAPK and PKC pathways in ISMFs . P62937 is required for efficient human cytomegalovirus DNA replication and reactivation . Human cytomegalovirus ( HCMV ) is a large DNA virus belonging to the subfamily Betaherpesvirinae . Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV . However , following terminal differentiation of these cells , virus is reactivated , and in an immunocompromised host acute infection can occur . It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections . P62937 ( CyPA ) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses , including human immunodeficiency virus , hepatitis C virus , murine cytomegalovirus , influenza A virus and vaccinia virus . This study investigated the role of CyPA during HCMV infection . CyPA expression was silenced in human foreskin fibroblast ( HF ) and THP-1 cells using small interfering RNA ( siRNA ) technology , or the cells were treated with cyclosporin A ( DB00091 ) to inhibit CyPA activity . Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early ( IE ) proteins , decreased viral DNA loads and reduced titres . Furthermore , silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state . Interestingly , it was observed that treatment of THP-1 cells with DB00091 prevented the cells from establishing a fully latent infection . In summary , these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells , and is a major component in virus reactivation from infected THP-1 cells . P62937 is required for Q9C035 {alpha}-mediated resistance to HIV-1 in Old World monkey cells . The peptidyl-prolyl isomerase cyclophilin A ( CypA ) embraces an exposed , proline-rich loop on HIV-1 capsid ( CA ) and renders reverse transcription complexes resistant to an antiviral activity in human cells . A CypA fusion with Q9C035 that is unique to New World owl monkeys also targets HIV-1 CA , but this interaction potently inhibits infection . A similar block to HIV-1 infection in Old World monkeys is attributable to the alpha isoform of the Q9C035 orthologue in these species . To determine whether HIV-1 restriction by Old World monkey TRIM5alpha is modulated by the CA-CypA interaction , RNA interference was used to disrupt CypA in cells from African green monkeys and rhesus macaques . HIV-1 infectivity increased in response to CypA knock-down to the same extent that it increased in response to Q9C035 knock-down . CypA knock-down eliminated the HIV-1 stimulatory effect of cyclosporin A ( DB00091 ) , a competitive inhibitor of the CypA-CA interaction , or of CA mutants that block binding to CypA but caused no change in titer of retroviruses that do n't interact with CypA . Simultaneous knock-down of both CypA and Q9C035 caused minimal additional increase in titer , suggesting that CypA inhibits HIV-1 replication in these cells because it is required for CA recognition by TRIM5alpha . Finally , DB00091 increased HIV-1 titer in otherwise nonrestrictive feline cells but only after these cells were transduced with Old World monkey TRIM5alpha . Thus , CypA is required for HIV-1 restriction by Old World monkey orthologues of TRIM5alpha . Evidences of monomer , dimer and trimer of recombinant human cyclophilin A . P62937 ( CyPA ) is a cytosolic receptor of immunosuppressive drug cyclosporin A ( DB00091 ) which possesses peptidyl-prodyl cis/trans isomerase ( PPIase ) activity . The recombinant human CyPA ( rhCyPA ) gene has been expressed in E. coli M15 . Purification was performed using salting-out , as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography . The molecular weight is about 18 kDa , confirmed by SDS-PAGE and mass spectrum . The results of Native-PAGE and immunoblotting showed the existence of three bands , which agreed well with the gel filtration results . The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE ( rhCyPA cross-linked with glutaraldehyde ) was 18 kDa , 36 kDa and 54 kDa respectively . Further more , the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration . All of the evidences above suggest that rhCyPA exists in forms of monomer , dimer and trimer . Moreover , we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution , and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer , which probably have some biological significances . Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype . P62937 ( CypA ) was originally identified as a cytosolic protein possessing peptidyl-prolyl isomerase activity . CypA has been shown to play a pivotal role in the immune response , but little is known about other molecular mechanisms of CypA-mediated biologic events . In our present study , we demonstrate that knockdown CypA expression using RNAi in U2OS cells resulted in disruption of the F-actin structure , as well as decreased anchorage-independent growth , proliferation , and migration . Wild-type U2OS cells treated with cyclosporine A ( DB00091 ) , a peptidyl-prolyl isomerase inhibitor , displayed the same phenotype as knockdown CypA cells , suggesting that the isomerase activity of CypA is required to maintain a normal phenotype . In vitro and in vivo binding assays revealed that CypA binds to O00401 , which functions in the nucleation of actin via the Arp2/3 complex . Pulse-chase labeling study indicated an enhanced degradation of O00401 in cell lacking CypA , suggesting that CypA is required for stabilizing O00401 to form a O00401 /Arp2/3 complex for the nucleation/initiation of F-actin polymerization . Concise prediction models of anticancer efficacy of 8 drugs using expression data from 12 selected genes . We developed concise , accurate prediction models of the in vitro activity for 8 anticancer drugs ( DB00544 , DB00515 , DB00305 , DOX , CPT-11 , SN-38 , TXL and TXT ) , along with individual clinical responses to DB00544 using expression data of 12 genes . We first performed cDNA microarray analysis and MTT assay of 19 human cancer cell lines to sort out genes which were correlative in expression levels with cytotoxicities of the 8 drugs ; we selected 13 genes with proven functional significance to drug sensitivity from a huge number of potent prediction marker genes . The correlation significance of each was confirmed using expression data quantified by real-time RT-PCR , and finally 12 genes ( P08183 , Q9UNQ0 , P10632 , P08684 , Q12882 , P09211 , P16455 , P15559 , P16435 , P11388 , P07437 and P04818 ) were selected as more reliable predictors of drug response . Using multiple regression analysis , we fixed 8 prediction formulae which embraced the variable expressions of the 12 genes and arranged them in order , to predict the efficacy of the drugs by referring to the value of Akaike 's information criterion for each sample . These formulae appeared to accurately predict the in vitro efficacy of the drugs . For the first clinical application model , we fixed prediction formulae for individual clinical response to DB00544 in the same way using 41 clinical samples obtained from 30 gastric cancer patients and found to be of predictive value in terms of survival , time to treatment failure and tumor growth . None of the 12 selected genes alone could predict such clinical responses . Conditional O00401 knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology . Cerebrospinal fluid ( P04141 ) is produced by the choroid plexus and moved by multi-ciliated ependymal cells through the ventricular system of the vertebrate brain . Defects in the ependymal layer functionality are a common cause of hydrocephalus . O00401 ( Neural-Wiskott Aldrich Syndrome Protein ) is a brain-enriched regulator of actin cytoskeleton and O00401 knockout caused embryonic lethality in mice with neural tube and cardiac abnormalities . To shed light on the role of O00401 in mouse brain development , we generated O00401 conditional knockout mouse model O00401 (fl/fl) ; P48681 -Cre ( NKO-Nes ) . NKO-Nes mice were born with Mendelian ratios but exhibited reduced growth characteristics compared to their littermates containing functional O00401 alleles . Importantly , all NKO-Nes mice developed cranial deformities due to excessive P04141 accumulation and did not survive past weaning . Coronal brain sections of these animals revealed dilated lateral ventricles , defects in ciliogenesis , loss of ependymal layer integrity , reduced thickness of cerebral cortex and aqueductal stenosis . Immunostaining for P19022 suggests that ependymal integrity in NKO-Nes mice is lost as compared to normal morphology in the wild-type controls . Moreover , scanning electron microscopy and immunofluorescence analyses of coronal brain sections with anti-acetylated tubulin antibodies revealed the absence of cilia in ventricular walls of NKO-Nes mice indicative of ciliogenesis defects . O00401 deficiency does not lead to altered expression of O00401 regulatory proteins , Fyn and Cdc42 , which have been previously implicated in hydrocephalus pathology . Taken together , our results suggest that O00401 plays a critical role in normal brain development and implicate actin cytoskeleton regulation as a vulnerable axis frequently deregulated in hydrocephalus . P62937 is required for P61073 -mediated nuclear export of heterogeneous nuclear ribonucleoprotein A2 , activation and nuclear translocation of P27361 /2 , and chemotactic cell migration . The chemokine receptor P61073 -mediated signaling cascades play an important role in cell proliferation and migration , but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood . Here , we demonstrate that P61073 was co-immunoprecipitated with cyclophilin A ( CyPA ) from the lysate of HEK293 cells stably expressing P61073 . Although both the glutathione S-transferase- P61073 N- and C-terminal fusion proteins were associated with the purified CyPA , truncation of the C-terminal domain of P61073 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells , thereby suggesting a critical role of the receptor C terminus in this interaction . Ligand stimulation of P61073 induced CyPA phosphorylation and nuclear translocation , both of which were inhibited by truncation of the C-terminal domain of P61073 . CyPA was associated with transportin 1 , and knockdown of transportin 1 by RNA interference ( RNAi ) blocked P48061 -induced nuclear translocation of CyPA , thereby suggesting a transportin 1-mediated nuclear import of CyPA . CyPA formed a complex with heterogeneous nuclear ribonucleoprotein ( hnRNP ) A2 , which underwent nuclear export in response to activation of P61073 . Interestingly , the P61073 -mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA . Moreover , P61073 -evoked activation of extracellular signal-regulated kinase 1/2 ( P27361 /2 ) was attenuated by CyPA RNAi , by overexpression of a PPIase-deficient mutant of CyPA ( CyPA-R55A ) , and by pretreatment of the immunosuppressive drugs , cyclosporine A and sanglifehrin A . Finally , P48061 -induced chemotaxis of HEK293 cells stably expressing P61073 or Jurkat T cells was inhibited by CyPA RNAi or DB00091 treatment . A new algorithm for weekly phenprocoumon dose variation in a southern Brazilian population : role for P11712 , P08684 /5 and Q9BQB6 genes polymorphisms . DB00946 is widely used in prophylaxis and treatment of thromboembolic disorders . However , its pharmacokinetics and pharmacodynamics vary according to several genetic and non-genetic factors . DB00946 metabolism is mediated by P11712 and CYP3A enzymes . Moreover , Q9BQB6 is phenprocoumon target of action . Therefore , the aim of this study was to evaluate the association of single nucleotide polymorphisms ( SNPs ) in Q9BQB6 , P11712 , P08684 and P20815 genes with the variance of weekly phenprocoumon dose as well as to develop an algorithm for dose prediction based on genetic and environmental factors . A total of 198 patients with stable phenprocoumon dose , 81 % of European ancestry , were investigated . Genotypes were determined by allelic discrimination with TaqMan assays . Polymorphisms -1639G > A and 1173C > T in Q9BQB6 and the presence of P11712 *2 and/or P11712 *3 are associated with lower doses . On the other hand , 3730G > A in Q9BQB6 gene is associated with higher doses . No association was found between P08684 *1B , P20815 *3 and P20815 *6 polymorphisms . Among non-genetic factors , gender , height , age and use of captopril , omeprazole , simvastatin and β-blockers are associated with dose . Two algorithms were derived : one for the whole sample explained 42 % of dose variation and one for patients of European ancestry only which explained 46 % of phenprocoumon dose . The mean absolute difference between observed and predicted dose was low in both models ( 3.92 mg/week and 3.54 mg/week , for models 1 and 2 , respectively ) . However , more studies with other genes and environmental factors are needed to test and to improve the algorithm . HCV NS5A and Q00978 compete for CypA binding . BACKGROUND & AIMS : P62937 ( CypA ) is vital for HCV replication . Cyp inhibitors successfully decrease viral loads in HCV-infected patients . However , their mechanisms of action remain unknown . Since interferon ( IFN ) can also suppress HCV replication , we asked whether a link between CypA and the IFN response exists . METHODS : We used cellular and recombinant pulldown approaches to investigate the possibility of a specific association of CypA with host ligands . RESULTS : We found for the first time that CypA binds to a major component of the IFN response - the IFN regulatory factor 9 ( Q00978 ) . Q00978 is the DNA-binding component of the transcriptional IFN-stimulated gene factor 3 ( ISGF3 ) . CypA binds directly to Q00978 via its peptidyl-prolyl isomerase ( PPIase ) pocket . Cyp inhibitors such as cyclosporine A ( DB00091 ) or non-immunosuppressive derivates such as alisporivir and SCY-635 , prevent Q00978 -CypA complex formation . CypA binds to the C-terminal Q969Q1 -association-domain ( IAD ) , but not to the DNA-binding or linker domains of Q00978 . Remarkably , CypA associates with the multimeric ISGF3 complex . We also obtained evidence that CypA neutralization enhances IFN-induced transcription . Interestingly , the hepatitis C virus ( HCV ) non-structural 5A ( NS5A ) protein , which is known to modulate the IFN response , competes with Q00978 for CypA binding and can prevent the formation of Q00978 -CypA complexes . CONCLUSIONS : This study demonstrates for the first time that CypA binds specifically to a component of the Janus kinase/signal transducer and activator of transcription ( JAK/ P35610 ) pathway , Q00978 . This study also reveals a novel opportunity of HCV to modulate the IFN response via NS5A . [ DB00707 sodium ( Photofrin-II ) ] . DB00707 sodium ( DB00707 ) is a photosensitizer which distributes selectively to tumor tissues , and causes tumor cell death by combination with light irradiation . Photodynamic therapy ( PDT ) by combination of porfimer sodium and laser was developed as a new cancer therapy . Tumor selectivity of porfimer sodium are based on the following reasons ; 1 ) high affinity for lipoprotein , especially , low density lipoprotein ( LDL ) , 2 ) elevation of P01130 activity in cancer tissue , and 3 ) lack or imcompleteness of lymphatic system in cancer tissue . DB00707 sodium is activated by laser irradiation at 630 nm , which can reacts with tissue oxygen to produce highly reactive excited siglet oxygen ( 1O2 ) . This highly reactive molecule is subsequently capable of killing tumor cells through oxidation of cellular component like mitochondrial enzymes . In addition , this highly reactive intermediate causes destruction of the tumor capillaries , which accelerates tumor cell death . The growth suppression or lethal damage to tumor cells by PDT of porfimer sodium and excimer dye laser were observed in experimental tumor models . In human clinical trials , the rates of complete response ( CR ) for roentgenographically occult lung cancer , stage I lung cancer , superficial esophageal cancer , superficial gastric cancer and carcinoma in situ or dysplasia of the cervix were 84.8 % , 50.0 % , 90.0 % , 87.5 % and 94.4 % , respectively . The major side effects were cutaneous symptoms e.g. photosensitivity , pigmentation , increasing GOT , GPT but these symptoms were not severe . PDT using porfimer sodium and excimer dye laser must be clinically useful for the treatment of inoperable early cancer or conservation of organ functions . Proteomic analysis identifies differentially expressed proteins in Q01196 / Q06455 acute myeloid leukemia cells treated with P26358 inhibitors azacitidine and decitabine . Azacitidine and decitabine are DNA methyltransferase inhibitors used to treat myelodysplastic syndromes and acute myeloid leukemias . To further characterize different mechanisms between these two agents , cellular extracts from leukemic cells untreated or treated with either drug were analyzed using 2D electrophoresis . Numerous differentially expressed proteins were identified with MALDI-TOF/TOF-MS . P62937 , P04040 , P06748 and P12004 were decreased exclusively by azacitidine , P17987 and P22626 by both drugs ; alpha-Enolase and Q06830 by decitabine . Interestingly , the expression of the proinflammatory protein P62937 , also suggested as marker of cell necrosis , was stimulated by decitabine . Finally , a comprehensive pathway analysis of data highlighted a relationship between the identified proteins and potential effectors . Discovering novel chemical inhibitors of human cyclophilin A : virtual screening , synthesis , and bioassay . P62937 ( CypA ) is a member of cyclophilins , a family of the highly homologous peptidyl prolyl cis-trans isomerases ( PPIases ) , which can bind to cyclosporin A ( DB00091 ) . CypA plays critical roles in various biological processes , including protein folding , assembly , transportation , regulation of neuron growth , and HIV replication . The discovery of CypA inhibitor is now of a great special interest in the treatment of immunological disorders . In this study , a series of novel small molecular CypA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay . The SPECS_1 database containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA . After SPR-based binding affinity assay , 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-molar level ( compounds 1-15 ) . Seven compounds were selected as the starting point for the further structure modification in considering binding activity , synthesis difficulty , and structure similarity . We thus synthesized 40 new small molecular compounds ( 1-6 , 15 , 16a-q , 17a-d , and 18a-l ) , and four of which ( compounds 16b , 16h , 16k , and 18g ) showed high CypA PPIase inhibition activities with IC50s of 2.5-6.2 microM . Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities against the proliferation of spleen cells . Somatostatin preserved blood brain barrier against cytokine induced alterations : possible role in multiple sclerosis . Multiple sclerosis ( MS ) is an inflammatory neurological disorder associated with demyelination , impaired blood brain barrier ( BBB ) , axonal damage and neuronal loss . In the present study , we measured somatostatin ( P61278 ) and tumor necrosis factor-α ( P01375 -α ) like immunoreactivity in P04141 samples from MS and non-MS patients . We also examined the role of P61278 in cytokines and lipopolysaccharide ( LPS ) -induced damage to the BBB using human brain endothelial cells in culture . Most of the cerebrospinal fluid ( P04141 ) samples studied from definite MS patients exhibited lower somatostatin ( P61278 ) -like immunoreactivity and higher expression of P01375 -α in comparison to non-MS patients . Treatment of cells with cytokines and LPS blocked P61278 secretion and decreased P61278 expression . Human brain endothelial cells expressed all five somatostatin receptors ( SSTRs ) with increased expression of P30874 and 4 upon treatment with cytokines and LPS . Cytokines and LPS-induced disruption of the tight junction proteins Zonula occludens ( ZO-1 ) organization was restored in presence of P61278 , P30874 or P31391 selective agonists . Furthermore , inflammation induced changes in extracellular signal-regulated kinases ( P27361 /2 and Q13164 ) signaling and altered expression of endothelial and inducible nitric oxide synthase are modulated in presence of P61278 . These data indicate that decreased levels of P61278 contribute to failure of the BBB in MS . Postprandial cell defense system responses to meal formulations : stratification through gene expression profiling . SCOPE : Cell defenses regulating homeostatic control of postprandial stress are influenced by interindividual variation , food composition and health status . This study investigates effects of food composition on individual postprandial responses and associations with health . METHODS AND RESULTS : Volunteers ( n = 16 ) consumed four food formulations ( 50 % unsaturated/saturated fat , with/without beetroot extract 10 g/100 g ) on separate occasions . GeXP assay measured whole blood postprandial gene expression profiles of 28 cell defense markers at baseline and postprandial time points 1 , 2 , 4 , 6 , 24 h . Plasma markers of metabolic lipids , hormones , inflammatory cytokines , oxidative stress , and DNA damage/repair were also assessed . SIRT 1 , P55851 , P09601 , P48637 , P35354 , P04637 , CDKN2A , P62937 , O14543 , and P27695 expression profiles revealed distinct stratified subgroups associated with plasma HDLs , P01375 -α and postprandial responses of O14543 , and P62937 . Leptin , P05231 , and DNA strand breaks revealed differing responses to fat type consumed . CONCLUSION : This study demonstrates postprandial immune , inflammatory , redox , metabolic , and DNA repair responses that are largely independent of fat type consumed ( unsaturated/saturated ) or addition of beetroot extract , in apparently healthy individuals . However , postprandial responses can be characterized by regulation of gene expression associated with markers linked to health status and are subject to interindividual variation that can influence postprandial responses . Glioma cell activation by Alzheimer 's peptide Abeta1-42 , alpha1-antichymotrypsin , and their mixture . We compared the effects ofAlzheimer 's peptide ( Abeta1-42 ) , a,-antichymotrypsin ( ACT ) and an ACT/Abeta1-42 mixture on human glioma DK-MG cells . The solution of Abeta ( 5 microM ) formed by 2-h incubation at room temperature induced tumour necrosis factor-alpha ( P01375 ) and interleukin ( IL ) -6 levels by 55 and 45 % , respectively , and increased gelatinase B activity by 67 % , while exposure of cells to the ACT/Abeta1-42 mixture ( 1:10 molar ratio ACT : Abeta1-42 ) under the same experimental conditions showed no effect on P05231 levels or gelatinase B activity , but strongly induced P01375 ( by 190 % ) , compared to the controls . Stimulation of the cells with Abeta1-42 alone , but not with ACT , increased by about 20 % low-density lipoprotein ( LDL ) uptake and mRNA levels for P01130 and P04035 , while the ACT/Abeta1-42 mixture significantly increased LDL uptake ( by 50 % ) , up-regulated mRNA levels for P01130 and P04035 by 48 and 63 % , respectively , and increased lipid accumulation by about 20-fold . These data suggest a possible new role for Abeta in Alzheimer 's disease through its interaction with the inflammatory reactant , ACT . Multiple pathways of apolipoprotein E signaling in primary neurons . P02649 is a genetic risk factor for Alzheimer 's disease , and the apoE protein is associated with beta-amyloid deposits in Alzheimer 's disease brain . We examined signaling pathways stimulated by apoE in primary neurons in culture . ApoE and an apoE-derived peptide activated several intracellular kinases , including prominently extracellular signal-regulated kinase 1/2 ( P27361 /2 ) . P27361 /2 activation by apoE was blocked by an inhibitor of the low-density lipoprotein receptor family , the specific DB01221 glutamate receptor antagonist MK 801 and other calcium channel blockers . Activation of apoE receptors also induced tyrosine phosphorylation of Dab1 , an adaptor protein of apoE receptors , but experiments in Dab1 knockout neurons demonstrated that Dab1 was not necessary for P29323 activation . In contrast , apoE treatment of primary neurons decreased activation of c-Jun N-terminal kinase , a kinase that interacts with another apoE receptor adaptor protein , c-Jun N-terminal kinase-interacting protein . This change also depended on interactions with the low-density lipoprotein receptor family but was independent of calcium channels . c-Jun N-terminal kinase deactivation by apoE was blocked by gamma-secretase inhibitors and pertussis toxin . These results demonstrate that apoE affects several signaling cascades in neurons : increased disabled phosphorylation , activation of the P27361 /2 pathway ( dependent on calcium influx via the DB01221 receptor ) and inhibition of the P45983 /2 pathway ( dependent on gamma-secretase and G proteins ) . Secreted cyclophilin A , a peptidylprolyl cis-trans isomerase , mediates matrix assembly of hensin , a protein implicated in epithelial differentiation . Q9UGM3 is a rabbit ortholog of Q9UGM3 , a multifunctional , multidomain protein implicated in the regulation of epithelial differentiation , innate immunity , and tumorigenesis . Q9UGM3 in the extracellular matrix ( Q13201 ) induced morphological changes characteristic of terminal differentiation in a clonal cell line ( clone C ) of rabbit kidney intercalated cells . Although hensin is secreted in monomeric and various oligomeric forms , only the polymerized Q13201 form is able to induce these phenotypic changes . Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase ( PPIase ) inhibitors cyclosporin A ( DB00091 ) and a derivative of cyclosporin A with modifications in the d- DB00133 side chain ( Cs9 ) but not by the calcineurin pathway inhibitor FK506 . PPIase inhibition led to failure of hensin polymerization in the medium and Q13201 , plus the loss of apical cytoskeleton , apical microvilli , and the columnar epithelial shape of clone C cells . P62937 was produced and secreted into the media to a much greater extent than cyclophilins B and C . Our results also identified the direct DB00091 -sensitive interaction of cyclophilin A with hensin , suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin . These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation . [ DB00091 inhibits the adhesion of neutrophil with ECV-304 induced by hypoxia/reoxygenation via ROS- P62937 - P27361 /2 pathway ] . To investigate the inhibition of cyclosporin A ( DB00091 ) on neutrophil adhesion to human umbilical vein endothelial cells ( HUVECs , ECV-304 ) induced by hypoxia/reoxygenation and further explore its mechanism , a 1 h hypoxia/4 h reoxygenation model was reproduced using ECV-304 . The adhesion rate of neutrophils to ECV-304 was determined by measuring the activity of endogenous hexosaminidase . The expression of endothelial cell adhesion molecules of P16581 and P05362 was measured by flow cytometry . The expression of cyclophilin A ( CyPA ) and the activation of P27361 /2 was compared among experimental groups by Western blot . The content of reactive oxygen species ( ROS ) was measured by Fenton reaction . After being stimulated with 1 h hypoxia/4 h reoxygenation , ECV-304 showed an enhanced neutrophil adhensiveness in association with an increased surface expression of P16581 and P05362 . In parallel , the content of ROS was also increased . These effects were significantly suppressed by the addition of DB00091 . Most importantly , the expression of CyPA was significantly increased following 1 h hypoxia/4 h reoxygenation , which was accompanied with an increased activation of P27361 /2 . Treatment with CyPA inhibitor DB00091 and CyPA antisense oligonucleotides significantly inhibited the activation of P27361 /2 and decreased the adhesion of neutrophils to ECV-304 . The specific P27361 /2 inhibitor PD98059 caused an inhibition of neutrophil adhesion to hypoxia/reoxygenation-stimulated ECV-304 . Our data confirm that DB00091 inhibits neutrophil adhesion to hypoxia/reoxygenation stimulated ECV-304 by a mechanism involving inhibition of the signal transduction of ROS , CyPA and P27361 /2 . Agonists of the Q9H244 -receptor activate Q96HU1 kinase by a ras-independent pathway in rat P13671 glioma . We have previously shown that an ecto-NPPase modulates the DB00171 - and ADP-mediated Q9H244 -receptor activation in rat P13671 glioma . In the present study , 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the Q9H244 -receptor with EC(50) values of 250 +/- 37 pM and 1 +/- 0.5 microM , respectively . Q9H244 -receptor stimulation increased Q96HU1 kinase ( P27361 /2 ) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the Q9H244 -purinoceptor . The purinoceptor antagonists DIDS and Q08999 blocked Q96HU1 kinase activation . An IP(3)-independent Ca(2+)-influx was observed after Q9H244 -receptor activation . Inhibition of this influx by Ca(2+)-chelation , did not affect Q96HU1 kinase activation . Pertussis toxin , toxin B , selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap(3)A-induced Q96HU1 kinase activation . In addition , transfection with dominant negative RhoA(Asn19) rendered P13671 cells insensitive to Q9H244 -receptor-mediated Q96HU1 kinase activation whereas dominant negative ras was without effect . Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after Q9H244 -receptor activation . We may conclude that Q9H244 -purinoceptor agonists activate Q96HU1 kinase through a G(i)-RhoA-PKC-raf-MEK-dependent , but ras- and Ca(2+)-independent cascade . The glucosylceramide synthase inhibitor PDMP sensitizes pancreatic cancer cells to MEK/ P29323 inhibitor AZD-6244 . Here we show that d,l-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( PDMP ) , a glycosphingolipid biosynthesis inhibitor , increases the sensitivity of pancreatic cancer cells to the novel MEK- P29323 inhibitor AZD-6244 . AZD-6244 and PDMP co-administration induced massive pancreatic cancer cell death and apoptosis , more potently than either drug alone . We discovered that AZD-6244 induced ceramide production in pancreatic cancer cells , yet the excess ceramide was metabolically removed in the long-term ( 24-48h ) . PDMP facilitated AZD-6244-induced ceramide production , and ceramide level remained elevated up to 48h . Meanwhile , exogenously-added cell-permeable short chain ceramide ( P06681 ) similarly sensitized AZD-6244 's activity , the two caused substantial pancreatic cancer cell death and apoptosis . At the molecular level , PDMP and AZD-6244 co-treatment inactivated P27361 /2 and AKT- P42345 signalings simultaneously in pancreatic cancer cells , while either agent alone only affected one signaling . In summary , PDMP significantly increased the sensitivity of AZD-6244 in pancreatic cancer cells . This appears to involve a sustained ceramide production as well as concurrent block of P29323 and AKT- P42345 signalings . A cyclophilin A inducible expressed in gonad of zhikong scallop Chlamys farreri . P62937 ( CypA ) , a receptor for the immunosuppressive agent cyclosporin A ( DB00091 ) , is a cis-trans peptidyl-prolyl isomerase ( PPIase ) which accelerates the cis-trans isomerization of prolyl-peptide bonds , interacts with a variety of proteins and therefore regulates their activities . One CypA ( designated CfCypA ) cDNA was cloned from Chlamys farreri by expressed sequence tag ( EST ) and rapid amplification of cDNA ends ( RACE ) techniques . The full-length cDNA of CfCypA consisted of 1,248 nucleotides with a canonical polyadenylation signal sequence AATAAA , a poly ( A ) tail , and an open reading frame ( ORF ) of 495 nucleotides encoding a polypeptide of 164 amino acids . The deduced amino acid sequence shared high similarity with CypA from the other species , indicating that CfCypA should be a new member of the CypA family . Quantitative real-time ( RT ) PCR was employed to assess the mRNA expression of CfCypA in various tissues and its temporal expression in haemocytes and gonad of scallops challenged with Vibrio anguillarum . The mRNA transcripts of CfCypA could be detected in all the examined tissues with highest expression level in gonad . After bacterial challenge , the expression level of CfCypA was almost unchanged in haemocytes , but up-regulated in gonad and increased to the peak ( 22.59-fold ; P < 0.05 ) at 4 h post-injection , and then dropped to the original level at 8 h post-injection . These results indicated that CfCypA was constitutive expressed in haemocytes , but could be induced in gonad , and perhaps played a critical role in response to the bacterial challenge in gonad . Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans . P62937 is the target of the immunosuppressant cyclosporin A ( DB00091 ) and is encoded by a single unique gene conserved from yeast to humans . In the pathogenic fungus Cryptococcus neoformans , two homologous linked genes , P15085 and P48052 , were found to encode two conserved cyclophilin A proteins . In contrast to Saccharomyces cerevisiae , in which cyclophilin A mutations confer DB00091 resistance but few other phenotypes , cyclophilin A mutations conferred dramatic phenotypes in C. neoformans . The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth , mating , virulence and DB00091 toxicity . The Cpa1 and Cpa2 proteins also have divergent functions . cpa1 mutants are inviable at 39 degrees C and attenuated for virulence , whereas cpa2 mutants are viable at 39 degrees C and fully virulent . cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence . P62937 active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures , suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function . DB00502 induces neurotoxicity by the DB01221 receptor downstream signaling pathway , alternative from glutamate excitotoxicity . The DB01221 receptor is believed to be important in a wide range of nervous system functions including neuronal migration , synapse formation , learning and memory . In addition , it is involved in excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders . Besides of agonist/coagonist sites , other modulator sites , including butyrophenone site may regulate the N-methyl-D-aspartate receptor . It has been shown that haloperidol , an antipsychotic neuroleptic drug , interacts with the Q13224 subunit of DB01221 receptor and inhibits DB01221 response in neuronal cells . We found that DB01221 receptor was co-immunoprecipitated by anti-Ras antibody and this complex , beside NR2 subunit of DB01221 receptor contained haloperidol-binding proteins , P29475 and Ras- P01286 . Furthermore , we have shown that haloperidol induces neurotoxicity of neuronal cells via DB01221 receptor complex , accompanied by dissociation of Ras- P01286 from membranes and activation of c-Jun-kinase . Inclusion of insulin prevented relocalization of Ras- P01286 and subsequent neuronal death . DB00502 -induced dissociation of Ras- P01286 leads to inhibition of membrane-bound form of Ras protein and changes downstream regulators activity that results in the initiation of the apoptotic processes via the mitochondrial way . Our results suggest that haloperidol induces neuronal cell death by the interaction with DB01221 receptor , but through the alternative from glutamate excitotoxicity signaling pathway . P62937 is upregulated in small cell lung cancer and activates P27361 /2 signal . P62937 ( CypA ) , a peptidyl-prolyl cis-trans isomerase ( PPIase ) , was originally identified as the intracellular receptor for cyclosporin A ( DB00091 ) . Recently , correlations of CypA with tumor pathogenesis have been studied . Here , we studied the expression of CypA and its receptor CD147 in several kinds of lung cancer cells as well as a normal lung cell and found that in H446 cell , a kind of small cell lung cancer cell , the expression are the highest . The exogeneous CypA protein can substantially stimulate H446 cell growth in dependence on its PPIase activity . We also showed that CypA protein can stimulate P27361 /2 signal in dose and time dependent manners and almost has no effect to p38 and JNK signals . Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for small cell lung cancer . Host cell species-specific effect of cyclosporine A on simian immunodeficiency virus replication . BACKGROUND : An understanding of host cell factors that affect viral replication contributes to elucidation of the mechanism for determination of viral tropism . P62937 ( CypA ) , a peptidyl-prolyl cis-trans isomerase ( PPIase ) , is a host factor essential for efficient replication of human immunodeficiency virus type 1 ( HIV-1 ) in human cells . However , the role of cyclophilins in simian immunodeficiency virus ( SIV ) replication has not been determined . In the present study , we examined the effect of cyclosporine A ( DB00091 ) , a PPIase inhibitor , on SIV replication . RESULTS : SIV replication in human CEM-SS T cells was not inhibited but rather enhanced by treatment with DB00091 , which inhibited HIV-1 replication . DB00091 treatment of target human cells enhanced an early step of SIV replication . CypA overexpression enhanced the early phase of HIV-1 but not SIV replication , while CypA knock-down resulted in suppression of HIV-1 but not SIV replication in CEM-SS cells , partially explaining different sensitivities of HIV-1 and SIV replication to DB00091 treatment . In contrast , DB00091 treatment inhibited SIV replication in macaque T cells ; DB00091 treatment of either virus producer or target cells resulted in suppression of SIV replication . SIV infection was enhanced by CypA overexpression in macaque target cells . CONCLUSIONS : DB00091 treatment enhanced SIV replication in human T cells but abrogated SIV replication in macaque T cells , implying a host cell species-specific effect of DB00091 on SIV replication . Further analyses indicated a positive effect of CypA on SIV infection into macaque but not into human T cells . These results suggest possible contribution of CypA to the determination of SIV tropism . 1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea : a novel cyclophilin A allosteric activator . P62937 ( CypA ) plays an important role in many physiology processes and its overexpression has been involved in many diseases including immune disease , viral infection , neuro-degenerative disease , and cancer . However , the actual role of CypA in the diseases is still far from clear , and a complete understanding of CypA is necessary in order to direct more specific and effective therapeutic strategies . Based on the screening of our in-house library through the isomer-specific proteolysis method , we find a CypA activator ( 1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea ) , compound 1a , which can increase CypA 's PPIase activity and give allosteric behavior . The binding affinity of compound 1a to CypA has been confirmed by Fortebio 's Octet Q13123 system and the increased phosphorylation of P29323 in H446 cells is observed by treatment with both compound 1a and DB00091 . In order to further evaluate the binding mode between the activator and CypA , the allosteric binding site and allosteric mechanism of CypA are investigated by molecular dynamics ( MD ) simulations in combination with mutagenesis experiments . The results show that the allosteric binding site of CypA is 7Å away from its catalytic site and is composed of Cys52 , His70 , His54 , Lys151 , Thr152 and Lys155 . Compound 1a binds to the allosteric site of CypA , stabilizing the active conformation of catalytic residues , and finally promotes the catalytic efficiency of CypA . We believe our finding of the CypA allosteric activator will be used as an effective chemical tool for further studies of CypA mechanisms in diseases . Genetic markers in the EET metabolic pathway are associated with outcomes in patients with aneurysmal subarachnoid hemorrhage . Preclinical studies show that epoxyeicosatrienoic acids ( EETs ) regulate cerebrovascular tone and protect against cerebral ischemia . We investigated the relationship between polymorphic genes involved in EET biosynthesis/metabolism , cytochrome P450 ( CYP ) eicosanoid levels , and outcomes in 363 patients with aneurysmal subarachnoid hemorrhage ( aSAH ) . Epoxyeicosatrienoic acids and dihydroxyeicosatetraenoic acid ( DHET ) cerebrospinal fluid ( P04141 ) levels , as well as acute outcomes defined by delayed cerebral ischemia ( P42126 ) or clinical neurologic deterioration ( CND ) , were assessed over 14 days . Long-term outcomes were defined by Modified Rankin Scale ( P59665 ) at 3 and 12 months . P10632 *4 allele carriers had 44 % and 36 % lower mean EET and DHET P04141 levels ( P=0.003 and P=0.007 ) and were 2.2- and 2.5-fold more likely to develop P42126 and CND ( P=0.039 and P=0.041 ) , respectively . P34913 55Arg , P51589 *7 , P10632 *1B , and P10632 g.36785A allele carriers had lower EET and DHET P04141 levels . P10632 g.25369T and P10632 g.36755A allele carriers had higher EET levels . Patients with P10632 *2C and P34913 404del variants had worse long-term outcomes while those with P34913 287Gln , P51589 *7 , and P11712 g.816G variants had favorable outcomes . Epoxyeicosatrienoic acid levels were associated with Fisher grade and unfavorable 3-month outcomes . Dihydroxyeicosatetraenoic acids were not associated with outcomes . No associations passed Bonferroni multiple testing correction . These are the first clinical data demonstrating the association between the EET biosynthesis/metabolic pathway and the pathophysiology of aSAH . Quantitative evaluation of reference genes for real-time PCR during in vitro maturation of ovine oocytes . Quantitative reverse transcription PCR ( RT-qPCR ) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre-implantation embryos . However , the high sensitivity of this technique requires that to prevent bias in expression data interpretation , the reference genes used for normalization are fully validated before use . Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte . In the present study , we evaluated eight commonly used reference genes such as P60709 , P04406 , P0C0S5 , P00492 , P62937 , P31040 , P07437 and P63104 and for sheep oocyte RT-qPCR before and after in vitro maturation . We have also compared different cDNA priming strategies using random hexamers or oligo-dT . GeNorm analysis of the results identified the most reliable genes for normalization to be P31040 , P07437 and P62937 when oocyte cDNA was made with random hexamers , and P63104 , P07437 and P31040 when oligo-dT primers were used ( P0C0S5 and P00492 were excluded from the geNorm analysis ) . Interestingly , the analysis revealed that the least stable genes were P60709 and P04406 , which are the conventional ' housekeeping ' genes used in many studies . We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the cDNA priming strategy employed . Contact sensitization to oxazolone : involvement of both interferon-gamma and interleukin-4 in oxazolone-specific Ig and T-cell responses . The synthesis and role of several lymphokines were examined during contact sensitization to oxazolone ( OX ) . Application of OX to the skin of mice increased the delayed-type hypersensitivity ( DTH ) response to challenge , serum titres of OX-specific IgG1 and IgG2a , and draining lymph node cell ( LNC ) numbers . At day 3 , LN contained detectable interleukin-4 ( P05112 ) , interferon-gamma ( P01579 ) and granulocyte-macrophage colony-stimulating factor ( GM- P04141 ) but not P60568 or P08700 mRNAs ; P08700 and higher levels of P05112 , P01579 and GM- P04141 mRNAs were measured after 24 hr culture with anti-CD3 antibody in OX-primed but not unprimed LNC . As a result of sensitization , LNC secreted P08700 constitutively and produced elevated levels of P60568 , P08700 , P05112 and P01579 in response to anti-CD3 antibody ; a similar but weaker lymphokine response was recalled by OX-protein conjugate . P01730 + cells were the major source of the anti-CD3-induced lymphokines except P01579 , which was derived mainly from CD8+ cells . Since both P05112 and P01579 were synthesized by OX-primed LNC in vivo and in vitro , their role was investigated by administering anti-lymphokine antibodies at the time of sensitization . Anti- P05112 treatment reduced OX-specific serum IgG1 titres without affecting IgG2a titres , whereas anti- P01579 treatment reduced IgG2a but not IgG1 titres . Although neither antibody altered DTH responsiveness , anti- P01579 treatment markedly increased P05112 production by P01730 + LNC and reduced P01579 production in vitro , particularly by P01730 + cells . We conclude that endogenous P05112 and P01579 reciprocally influence the isotype of the Ig response to OX and that P01579 also affects the relative levels of P05112 and P01579 synthesis by P01730 + LNC . Prasugrel : a new antiplatelet drug for the prevention and treatment of cardiovascular disease . Prasugrel , trade name DB06209 , is an investigational new antiplatelet drug currently under review for clinical use by the Food and Drug Administration . It is a thienopyridine analog with a structure similar to that of clopidogrel and ticlopidine . Thienopyridine derivatives inhibit platelet aggregation induced by adenosine diphosphate by irreversibly inhibiting the binding of adenosine diphosphate to the purinergic Q9H244 receptor on the platelet surface . Prasugrel has been shown to be a potent antiplatelet agent with a faster , more consistent , and greater inhibition of platelet aggregation compared with clopidogrel . It is debatable , however , how effectively these pharmacologic benefits will translate to clinical benefits . The results of the large TRITON-TIMI 38 trial , which compared prasugrel and clopidogrel in patients with acute coronary syndrome who were scheduled to receive coronary stents , demonstrated a significant reduction in ischemic events , including stent thrombosis , with prasugrel , but with an increased risk of major bleeding . The exact role of prasugrel in the management of ischemic heart disease is still being defined , but the risk:benefit ratio will likely play a major role in directing the best place for therapy with this new agent . Is phentermine an inhibitor of monoamine oxidase ? A critical appraisal . DB00191 produces a spectrum of concentration-dependent biochemical effects . It interacts with NE transporters at 0.1 microM , DA transporters at about 1 microM , 5-HT transporters at 15 microM and P21397 at about 100 microM . When administered at typical anorectic doses , phentermine primarily interacts with DA and NE transporters and does not produce biochemical or neurochemical effects which would occur if it were inhibiting P21397 . Some other explanation other than MAO inhibition must be sought to explain how oral phentermine increases platelet 5-HT , since platelet P27338 does not metabolize platelet 5-HT , and since amphetamine-type drugs are even weaker inhibitors of P27338 than P21397 . Clinical studies in humans have shown that amphetamine , which is a more potent inhibitor of P21397 than phentermine , does not inhibit P21397 at therapeutic doses . Neither phentermine alone , fluoxetine alone or their combined use have been associated with cardiac valvulopathy , and clinical experience has shown their combined use to be free of significant adverse effects . Viewed collectively , there appears to be no data to support the hypothesis that phentermine inhibits MAO at typical therapeutic doses . Use of a collagen biomatrix ( TissuDura ) for dura repair : a long-term neuroradiological and neuropathological evaluation . PURPOSE : The aim of this study was to evaluate the clinical , neuroradiological , and neuropathological outcomes of patients treated with equine collagen foil ( TissuDura ) as a dura mater substitute during cranial and spinal neurosurgical procedures . MATERIALS AND METHODS : All patients treated at the Department of Neurosurgery of the Second University of Naples with TissuDura between 2005 and 2009 were included . Dural reconstruction was performed using TissuDura , overlaid 1 cm over the dural defect with additional fixation using fibrin glue . No surgical sutures were used . Patients underwent postoperative contrast-enhanced magnetic resonance scans at 1 week , 1 month , and 1 year after surgery to detect any cerebrospinal fluid ( P04141 ) leaks , infections , inflammations , or P04141 circulation in the surgical region . RESULTS : Dural reconstruction was performed in 74 patients , including 50 patients with tumors , two with P06681 neurinoma , two with acoustic neurinoma , six with Chiari I malformation , two with severe head injury , and 12 requiring spinal surgery . Clinical and neuroradiological findings were normal and no signs of graft rejection or P04141 leaks at postoperative follow-up were observed . In two cases of atypical meningioma , re-operation of the dural reconstruction was performed after 1 year . No adherences between brain and neodura were detected , and histopathological investigations demonstrated dural regeneration . CONCLUSIONS : Following dural reconstructions with TissuDura without surgical sutures , no local toxicity or complications were observed for up to 1 year . TissuDura demonstrated elasticity , non-reactivity , and good adaptability . The overlay technique using fibrin glue was simple and fast . Future studies and longer follow-up are needed to confirm the efficacy of TissuDura . Comparative cytotoxicity of folate-based inhibitors of thymidylate synthase and 5-fluorouracil +/- leucovorin in MGH-U1 cells . P04818 ( TS ) is a critical enzyme in the synthesis of DNA and an important target for cancer chemotherapy . DB00544 ( 5FU ) combined with leucovorin ( LV ) has been used to inhibit TS , and inhibition is dependent on the formation of a ternary complex between a folate cofactor , TS , and 5-fluorodeoxyuridine monophosphate ( FdUMP ) , a metabolite of FU . The folate-based TS inhibitors CB3717 , its analogs , and BW1843U89 have been synthesized as specific inhibitors of TS that do not require activation or the presence of a cofactor . We have compared the cytotoxicity of 5FU +/- LV with that of these folate-based TS inhibitors in human bladder cancer MGH-U1 cells using a colony-forming assay . After a 6-h exposure , FU+LV , CB3717 , dCB3717 , or P06681 methyl dideazafolate analogs demonstrated similar cytotoxic potency that was 0.96 to 2.9 times that of 5FU alone . A 24-h exposure did not increase the potency of 5FU+LV relative to 5FU alone , but there was a marked increase in the cytotoxicity of the dideazafolates as compared with 5FU+LV . Similarly , BW1843U89 was more cytotoxic than 5FU/LV . This was reflected in a 3.2- to 1333-fold decrease in the 50 % inhibitory concentration ( IC50 ) . Simultaneous exposure to LV and thymidine ( TdR ) protected MGH-U1 cells from the cytotoxicity of CB3717 , its analogs , and BW1843U89 . We conclude that ( a ) the folate-based TS inhibitors are more potent than 5FU+LV after a 24-h exposure , ( b ) protection by LV and TdR indicates that TS inhibition is the primary site of action , and ( c ) BW1843U89 is more potent than D1694 in MGH-U1 cells . Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells . P62937 ( CypA ) is a peptidyl-prolyl isomerase that binds to the capsid protein ( CA ) of human immunodeficiency virus type 1 ( HIV-1 ) and by doing so facilitates HIV-1 replication . Although CypA is incorporated into HIV-1 virions by virtue of CypA-Gag interactions that occur during virion assembly , in this study we show that the CypA-CA interaction that occurs following the entry of the viral capsid into target cells is the major determinant of CypA 's effects on HIV-1 replication . Specifically , by using normal and CypA-deficient Jurkat cells , we demonstrate that the presence of CypA in the target and not the virus-producing cell enhances HIV-1 infectivity . Moreover , disruption of the CypA-CA interaction with cyclosporine A ( DB00091 ) inhibits HIV-1 infectivity only if the target cell expresses CypA . The effect of DB00091 on HIV-1 infection of human cells varies according to which particular cell line is used as a target , and CA mutations that confer DB00091 resistance and dependence exert their effects only if target cells , and not if virus-producing cells , are treated with DB00091 . The differential effects of DB00091 on HIV-1 infection in different human cells appear not to be caused by polymorphisms in the recently described retrovirus restriction factor TRIM5alpha . We speculate that CypA and/or CypA-related proteins affect the fate of incoming HIV-1 capsid either directly or by modulating interactions with unidentified host cell factors . Proof of concept study to assess fetal gene expression in amniotic fluid by nanoarray PCR . Microarray analysis of cell-free RNA in amniotic fluid ( AF ) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype . Once informative genes are identified , research moves to a more focused platform such as quantitative reverse transcriptase-PCR . Standardized NanoArray PCR ( P60880 ) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids . We used a previously developed P60880 gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant . RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation , and transcript abundance of 21 genes was measured . Statistically significant differences in expression , as a function of advancing gestational age , were observed for 5 of 21 genes . P08758 , P08236 , and P62937 showed decreasing gene expression over time , whereas O15234 and O43296 showed increasing gene expression over time . Statistically significantly increased expression of P42345 and P52630 was seen in female compared with male fetuses . This study demonstrates the feasibility of focused fetal gene expression analysis using P60880 technology . In the future , this technique could be optimized to examine specific genes instrumental in fetal organ system function , which could be a useful addition to prenatal care . P62937 facilitates translocation of the Clostridium botulinum P06681 toxin across membranes of acidified endosomes into the cytosol of mammalian cells . The binary Clostridium botulinum P06681 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I , which mono-ADP-ribosylates actin in eukaryotic cells . Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol . We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90 . Here , we demonstrate that cyclosporin A , which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins , inhibited intoxication of cells with P06681 toxin and prevented uptake of C2I into the cytosol . DB00091 blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes . In vitro , the addition of cytosol to P06681 toxin-loaded endosomes induced translocation of C2I activity into the cytosol , which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A . Pull-down experiments with lysates from P06681 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I . In conclusion , our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of P06681 toxin into mammalian cells . P62937 is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-deficient mice . P62937 ( CyPA ; encoded by Ppia ) is a ubiquitously expressed protein secreted in response to inflammatory stimuli . CyPA stimulates vascular smooth muscle cell migration and proliferation , endothelial cell adhesion molecule expression , and inflammatory cell chemotaxis . Given these activities , we hypothesized that CyPA would promote atherosclerosis . P02649 -deficient ( Apoe(-/-) ) mice fed a high-cholesterol diet for 16 wk developed more severe atherosclerosis compared with Apoe(-/-)Ppia(-/-) mice . Moreover , CyPA deficiency was associated with decreased low-density lipoprotein uptake , P19320 ( vascular cell adhesion molecule 1 ) expression , apoptosis , and increased P29474 ( endothelial nitric oxide synthase ) expression . To understand the vascular role of CyPA in atherosclerosis development , bone marrow ( BM ) cell transplantation was performed . Atherosclerosis was greater in Apoe(-/-) mice compared with Apoe(-/-)Ppia(-/-) mice after reconstitution with CyPA(+/+) BM cells , indicating that vascular-derived CyPA plays a crucial role in the progression of atherosclerosis . These data define a role for CyPA in atherosclerosis and suggest CyPA as a target for cardiovascular therapies . Naive P01730 t cell proliferation is controlled by mammalian target of rapamycin regulation of Q8TEB7 expression . In this study , we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes ( Q8TEB7 ) is expressed in quiescent naive mouse and human P01730 T cells and has a functional role in inhibiting naive T cell proliferation . Following TCR engagement , P10747 costimulation results in the expression of P60568 whose signaling through its receptor activates the Akt-mammalian target of rapamycin ( P42345 ) pathway . Activation of P42345 allows selective mRNA translation , including the epistatic regulator of Q8TEB7 , Q96FW1 ( Otub1 ) , whose expression results in the degradation of Q8TEB7 and allows T cell proliferation . The activation of P42345 appears to be the critical component of IL-2R signaling regulating Q8TEB7 expression . P16410 -Ig treatment blocks P10747 costimulation and resultant P60568 expression , whereas rapamycin and anti- P60568 treatment block P42345 activation downstream of IL-2R signaling . Thus , all three of these biotherapeutics inhibit P42345 -dependent translation of mRNA transcripts , resulting in blockade of Otub1 expression , maintenance of Q8TEB7 , and inhibition of P01730 T cell proliferation . These observations provide a mechanistic pathway sequentially linking P10747 costimulation , IL-2R signaling , and P42345 activation as important requirements for naive P01730 T cell proliferation through the regulation of Otub1 and Q8TEB7 expression . Our findings also extend the role of Q8TEB7 beyond anergy induction and maintenance , suggesting that endogenous Q8TEB7 regulates general cell cycle and proliferation of primary naive P01730 T cells .