interacts_with DB00642?
Translational research with pemetrexed in breast cancer . DB00642 ( Alimta ) is a novel folate antimetabolite that primarily inhibits the enzymes thymidylate synthase ( TS ) , dihydrofolate reductase ( P00374 ) , and glycinamide ribonucleotide formyl transferase ( GARFT ) , all of which are involved in pyrimidine and purine synthesis . In a phase II trial of patients with DB00279 /4 , N0-2 breast cancer , expression of thymidylate synthase ( TS ) , dihydrofolate reductase ( P00374 ) , glycinamide ribonucleotide formyltransferase ( GARFT ) , p53 , and c-erb-B2 ( at the mRNA or protein level ) was examined in tumor biopsy specimens before and 24 hours after the first dose of pemetrexed and after three cycles of single-agent treatment to establish correlations of biomarker levels and changes with clinical outcome and toxicity . Although final data are not available , initial indications are that clinical response may correlate with decreased or low TS expression . The results obtained from clinical data are supported by laboratory results in three cell lines ( MDA-231 , MCF-7 , and ZR-75 ) . These results suggest that in vitro transcript profiling to identify which genes are important predictors of successful cytotoxic chemotherapy , followed by a focused clinical trial to confirm the in vitro results , may be the best approach for translational research . An improved effect size for single-case research : nonoverlap of all pairs . Nonoverlap of All Pairs ( Q8WYA6 ) , an index of data overlap between phases in single-case research , is demonstrated and field tested with 200 published AB contrasts . Q8WYA6 is a novel application of an established effect size known in various forms as Area Under the Curve ( AUC ) , the Common Language Effect Size ( CL ) , the Probability of Superiority ( PS ) , the Dominance Statistic ( DS ) , Mann-Whitney 's U , and Sommers D , among others . Q8WYA6 was compared with 3 other non-overlap-based indices : P01160 ( percent of nonoverlapping data ) , P15941 ( percent of data points exceeding the median ) , and PAND ( percent of all nonoverlapping data ) , as well as Pearson 's R(2) . Five questions were addressed about Q8WYA6 : ( a ) typical Q8WYA6 values , ( b ) its ability to discriminate among typical single-case research results , ( c ) its power and precision ( confidence interval width ) , ( d ) its correlation with the established effect size index , R(2) , and ( e ) its relationship with visual judgments . Results were positive , the new index equaling or outperforming the other overlap indices on most criteria . DB00642 alters folate phenotype and inflammatory profile in EA.hy 926 cells grown under low-folate conditions . Elevated homocysteine is a risk marker for several major human pathologies . Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators . DB00642 acts by inhibiting thymidylate synthetase ( P04818 ) , dihydrofolate reductase ( P00374 ) , and glycinamide ribonucleotide formyltransferase ( GARFT ) . EA.hy 926 cells grown under low ( " Lo " ) and high ( " Hi " ) folate conditions were treated with pemetrexed . The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS . Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells . Treatment with pemetrexed caused a decrease in individual folate analytes . Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells . Several of the significantly up-regulated transcripts were inflammatory . Changes in transcript levels of selected targets , including P01024 , P10145 , and P00374 , were confirmed by quantitative RT-PCR . P01024 and P10145 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls ; P00374 transcript levels were decreased . In Lo cells , P10145 and P01024 protein concentrations were increased following pemetrexed treatment . DB00642 drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells . No such changes were observed in Hi cells , suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency . DB00642 : its promise in treating non-small-cell lung cancer . The use of chemotherapy in the treatment of early and advanced non-small-cell lung cancer ( NSCLC ) has increased during the past decade . One of the main reasons for the increased acceptance of chemotherapy is the development of several new cytotoxic agents with a unique mechanism(s) of action and high single-agent activity , combined with a favorable toxicity profile . DB00642 ( Alimta ) is a novel antifolate that inhibits several enzymes involved in DNA synthesis ( thymidylate synthase [ TS ] , dihydrofolate reductase [ P00374 ] , and glycinamide ribonucleotideformyltransferase [ GARFT ] ) . DB00642 's toxicity is markedly reduced by folic acid and vitamin B12 supplementation . The compound has been studied extensively in various tumor types , including NSCLC . In NSCLC , pemetrexed at 500 mg/m2 , every 3 weeks , given i.v. over 10 minutes , has shown promising activity , and can safely be administrated with vitamin supplementation . After registration , single-agent pemetrexed will certainly add to the chemotherapeutic options available for pretreated patients and will most likely change significantly chemotherapy prescriptions in second-line chemotherapy . In first-line chemotherapy , the role of platinum-based and -free combination doublet chemotherapy with pemetrexed still needs to be defined . Phase II data indicate high efficacy combined with favorable toxicity for pemetrexed in combination with cisplatin , carboplatin ( DB00958 ) , oxaliplatin ( Eloxatin ) , gemcitabine ( Gemzar ) , and vinorelbine ( Navelbine ) . This review summarizes the clinical experience obtained thus far during the early clinical development of pemetrexed in NSCLC . P42892 and β-arrestins exert spatiotemporal control of DB05875 -induced inflammatory signals . Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events , it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation . By using bioluminescence resonance energy transfer and superresolution microscopy , we found that DB05875 ( SP ) induces the association of the neurokinin 1 receptor ( P25103 ) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes : the scaffolding proteins β-arrestin ( βARRs ) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d . In HEK293 cells and non-transformed human colonocytes , we observed that G protein-coupled receptor kinase 2 and β P49407 /2 terminate plasma membrane Ca(2+) signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus . βARRs deliver the SP- P25103 endosomes , where P42892 associates with the complex , degrades SP , and allows the P25103 , freed from βARRs , to recycle . Thus , both P42892 and βARRs mediate the resensitization of P25103 Ca(2+) signaling at the plasma membrane . Sustained exposure of colonocytes to SP activates NF-κB and stimulates P10145 secretion . This proinflammatory signaling is unaffected by inhibition of the endosomal P29323 pathway but is suppressed by P42892 inhibition or β P32121 knockdown . Inhibition of protein phosphatase 2A , which also contributes to sustained P25103 signaling at the plasma membrane , similarly attenuates P10145 secretion . Thus , the primary function of βARRs and P42892 in SP-dependent inflammatory signaling is to promote resensitization , which allows the sustained P25103 signaling from the plasma membrane that drives inflammation . DB00642 : mRNA expression of the target genes TS , GARFT and P00374 correlates with the in vitro chemosensitivity of human solid tumors . Activation of the MyD88 signaling pathway inhibits ischemia-reperfusion injury in the small intestine . Toll-like receptors ( TLRs ) recognize microbial components and trigger the signaling cascade that activates innate and adaptive immunity . Recent studies have shown that the activation of TLR-dependent signaling pathways plays important roles in the pathogenesis of ischemia-reperfusion ( I/R ) injuries in many organs . All TLRs , except O15455 , use a common adaptor protein , MyD88 , to transduce activation signals . We investigated the role of MyD88 in I/R injury of the small intestine . MyD88 and cyclooxygenase-2 ( P35354 ) knockout and wild-type mice were subjected to intestinal I/R injury . I/R-induced small intestinal injury was characterized by infiltration of inflammatory cells , disruption of the mucosal epithelium , destruction of villi , and increases in myeloperoxidase activity and mRNA levels of P01375 -α and the P10145 homolog KC . MyD88 deficiency worsened the severity of I/R injury , as assessed using the histological grading system , measuring luminal contents of hemoglobin ( a marker of intestinal bleeding ) , and counting apoptotic epithelial cells , while it inhibited the increase in mRNA expression of P01375 -α and KC . I/R significantly enhanced P35354 expression and increased PGE(2) concentration in the small intestine of wild-type mice , which were markedly inhibited by MyD88 deficiency . P35354 knockout mice were also highly susceptible to intestinal I/R injury . Exogenous PGE(2) reduced the severity of injury in both MyD88 and P35354 knockout mice to the level of wild-type mice . These findings suggest that the MyD88 signaling pathway may inhibit I/R injury in the small intestine by inducing P35354 expression . Establishment of pemetrexed-resistant non-small cell lung cancer cell lines . DB00642 ( P15941 ) , a multitargeted antifolate with manageable toxicity , is active against non-squamous non-small cell lung cancer ; however , most patients eventually acquire resistance to P15941 . To elucidate the resistant mechanism , we established P15941 -resistant lung adenocarcinoma cell lines . Two parental cell lines , PC-9 and A549 , were treated with step-wise increasing concentrations of P15941 . Growth inhibition was determined by the 3-[4,5-dimethyl-thizol-2-yl]-2,5-diphenyltetrazolium bromide assay . Expression of the genes encoding thymidylate synthase ( TS ) , dihydrofolate reductase ( P00374 ) , and glycinamide ribonucleotide formyltransferase ( GARFT ) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction . The four PC-9 sublines were more resistant than the PC-9 cell line to P15941 ( 2.2- , 2.9- , 8.4- , and 14.3-fold , respectively ) . The four A549 sublines also showed more resistance to P15941 ( 7.8- , 9.6- , 42.3- , and 42.4-fold , respectively ) than the parent cell line . All resistant sublines showed cross-resistance to cisplatin , but not to docetaxel , vinorelbine , 5-fluorouracil , or the active metabolite of irinotecan , SN-38 . All P15941 -resistant sublines expressed more TS than the parental cells , by polymerase chain reaction and Western blotting . P00374 was significantly increased in the four P15941 -resistant A549 sublines . GARFT did not correlate with resistance to P15941 . In summary , P15941 -resistant cells remained sensitive to docetaxel , vinorelbine , 5-fluorouracil , and irinotecan . TS expression appeared to be associated with resistance to P15941 . P13569 in a lipid raft- P19438 complex modulates gap junctional intercellular communication and P10145 secretion . Mutations in the cystic fibrosis transmembrane conductance regulator ( P13569 ) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis ( CF ) . We have showed that P01375 signaling through the Src family tyrosine kinases ( SFKs ) was defective as determined by an inability of P01375 to regulate gap junctional communication ( GJIC ) in CF cells . Here , we sought to elucidate the mechanisms linking P01375 signaling to the functions of P13569 at the molecular level . In a MDCKI epithelial cell model expressing wild-type ( WtCFTR ) or mutant P13569 lacking its PDZ-interacting motif ( P13569 -DeltaTRL ) , P01375 increased the amount of WtCFTR but not P13569 -DeltaTRL in detergent-resistant membrane microdomains ( DRMs ) . This recruitment was modulated by SFK activity and associated with O60565 localization of P19438 and c-Src . Activation of P19438 signaling also decreased GJIC and markedly stimulated P10145 production in WtCFTR cells . In contrast , the absence of P13569 in DRMs was associated with abnormal P19438 signaling as revealed by no recruitment of P19438 and c-Src to lipid rafts in P13569 -DeltaTRL cells and loss of regulation of GJIC and P10145 secretion . These results suggest that localization of P13569 in lipid rafts in association with c-Src and P19438 provides a responsive signaling complex to regulate GJIC and cytokine signaling . Efficacy and safety of repeated dosing of netupitant , a neurokinin-1 receptor antagonist , in treating overactive bladder . AIM : NK-1 receptors in sensory nerves , the spinal cord and bladder smooth muscle participate in complex sensory mechanisms that regulate bladder activity . This study was designed to assess the efficacy and safety of a new P25103 antagonist , netupitant , in patients with OAB . METHODS : This was a phase II , multicenter , double-blind study in which adults with OAB symptoms > 6 months were randomized to receive 1 of 3 doses of netupitant ( 50 , 100 , 200 mg ) or placebo once daily for 8 weeks . The primary efficacy endpoint was percentage change from baseline in average number of daily micturitions at week 8 . Urinary incontinence , urge urinary incontinence ( UUI ) , and urgency episodes were also assessed . RESULTS : The primary efficacy endpoint was similar in the treatment groups ( -13.85 for placebo to -16.17 in the netupitant 200 mg group ) with no statistically significant differences between netupitant and placebo . The same was true for most secondary endpoints although a significant difference for improvement in UUI episodes and a trend for the greatest decrease in urgency episodes were seen in the netupitant 100 mg group . DB09048 was well tolerated with most treatment emergent adverse events ( AEs ) being mild . While the overall incidence of AEs increased with netupitant dose , there was no evidence for this dose dependency based on relationship to treatment , intensity , or time to onset . CONCLUSIONS : The study failed to demonstrate superiority of netupitant versus placebo in decreasing OAB symptoms , despite a trend favoring netupitant 100 mg . There were no safety concerns with daily administration of netupitant over 8 weeks . Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells . P13569 ( P13569 ) is a P13569 in epithelial cells ; recently , we identified it in mast cells . Previous work that we confirmed showed that interferon gamma ( IFNgamma ) down-regulated P13569 expression in epithelial cells ( T84 ) , but by contrast , we found that IFNgamma up-regulated P13569 mRNA and protein expression in rat and human mast cells . IFNgamma up-regulation of P13569 in mast cells was inhibited by p38 and extracellular signal-regulated kinase ( P29323 ) kinase inhibitors but not a Janus tyrosine kinase (JAK)2 inhibitor , whereas in T84 cells IFNgamma-mediated down-regulation of P13569 was O60674 -dependent and P29323 - and p38-independent . Furthermore , IFNgamma down-regulation of P13569 in T84 epithelial cells was P42224 -dependent , but up-regulation of P13569 in mast cells was P42224 -independent . Thus , differential regulatory pathways of P13569 expression in mast cells and epithelial cells exist that depend upon either p38/ P29323 or JAK/ P35610 pathways , respectively . Surprisingly , IFNgamma treatment of mast cells inhibited Cl(-) efflux , in contrast to up-regulation of P13569 /mRNA and protein expression . However , down-regulation of Cl(-) flux correlated with IFNgamma-mediated inhibition of mediator secretion . This and other work suggests that the effect of IFNgamma on P13569 expression in mast cells is important for their function . Mucin variable number tandem repeat polymorphisms and severity of cystic fibrosis lung disease : significant association with P98088 . Variability in cystic fibrosis ( CF ) lung disease is partially due to non- P13569 genetic modifiers . Mucin genes are very polymorphic , and mucins play a key role in the pathogenesis of CF lung disease ; therefore , mucin genes are strong candidates as genetic modifiers . DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat ( VNTR ) length polymorphisms for P15941 , Q02817 , P98088 , and Q8TAX7 . VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium ( LD ) with flanking single nucleotide polymorphisms ( SNPs ) . No strong associations were found for P15941 , Q02817 , or Q8TAX7 . A significant association was found between the overall distribution of P98088 VNTR length and CF lung disease severity ( p = 0.025 ; n = 468 patients ) ; plus , there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease ( p = 6.2×10(-4) after Bonferroni correction ) . There was strong LD between P98088 VNTR length modes and flanking SNPs . The severity-associated 6.4 kb VNTR allele of P98088 was confirmed to be genetically distinct from the 6.3 kb allele , as it showed significantly stronger association with nearby SNPs . These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients . Our data also show a novel link between the P98088 6.4 kb VNTR allele and severity of CF lung disease . The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains , or is linked to , important functional genetic variation . The role of pemetrexed in advanced non small-cell lung cancer : special focus on pharmacology and mechanism of action . DB00642 is a newer antifolate drug that has been approved as first-line treatment for patients with advanced non-squamous , non-small cell lung cancer ( NSCLC ) in combination with cisplatin , and as single agent for relapsed or chemotherapy refractory NSCLC after platinum-containing chemotherapy , at a dose of 500 mg/m(2) . DB00642 undergoes intracellular activation by poly-gamma-glutamylation , that is essential for its antiproliferative activity . Polyglutamate derivatives mainly inhibit three key enzymes of intracellular folate metabolism , i.e. thymidylates synthase ( P04818 ) , dihydrofolate reductase ( P00374 ) , and glycinamide ribonucleotide formyltransferase ( GARFT ) , with P04818 being the most relevant target . DB00642 undergoes rapid renal elimination as unchanged parent compound , with a terminal half-life of between two to five hours . In later clinical development , the usefulness of supplementation with folic acid and vitamin B(12) became evident , to control pemetrexed-related toxicity . The results from the phase III upfront registration study , a retrospective observational data , and a recent maintenance study of pemetrexed in NSCLC suggest histological subtype to be the most important predictive marker for clinical outcome in patients receiving pemetrexed , DB00642 is active in patients with non-squamous cell NSCLC while no benefit is seen in patients with squamous-cell histology , possibly as a result of different expression of intratumoral P04818 . These are important steps towards individualisation of anticancer treatment in patients with advanced NSCLC . Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes . Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge . This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels . Using whole-cell recordings , we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge . These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions : I- > NO3- > Cl- > Gluc- > or = MeS- > Ise- . Interestingly , extracellular I- , albeit showing the greatest permeability , blocked the currents with an IC50 of approximately 50 mM . Currents were almost completely inhibited by 123 microM P16860 and partially inhibited by 200 microM niflumic acid or 200 microM DIDS . Additionally , the total number of Cl- ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation . We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes . To examine potential candidate chloride channel genes expressed by astrocytes , we employed RT-PCR to demonstrate the presence of transcripts for P51788 , 3 , 4 , 5 , and 7 , as well as for P21796 and P13569 in cultured astrocytes . Moreover , we performed immunostaining with antibodies against each of these channels and showed the strongest expression of P51788 and P51790 , strong expression of P51795 and P21796 , weak expression of P51798 and very weak expression of P51793 and P13569 . Intriguingly , although we found at least seven Cl- channel proteins from three different gene families in astrocytes , none appeared to be active in resting cells . [ Role of neurokinin-1 receptor in lung injury in rats with acute necrotizing pancreatitis ] . OBJECTIVE : To investigate the expression of neurokinin-1 receptor ( P25103 ) in the lung tissue , and the relationship between expression of P25103 and lung injury in rats with acute necrotizing pancreatitis ( P01160 ) . METHODS : One hundred and twenty adult Sprague-Dawley rats were randomly divided into P01160 and control groups . Animals in group P01160 were induced by the retrograde intraductal infusion of 5 % sodium taurocholate ( 0.1 ml/kg ) , and animals in normal control group received laparotomy only . The accumulation of polymorphonuclear leukocytes in lung tissues was measured with myeloperoxidase ( P05164 ) assay . Lung endothelial barrier destruction was measured by lung capillary permeability ( LCP ) . Reverse transcription polymerase chain reaction ( RT-PCR ) was used to determine the mRNA expression of P25103 , western blot analysis was used to determine P25103 protein expression levels , and immunohistochemistry was used to localize expression site of P25103 . RESULTS : P25103 mRNA level was enhanced in the lung of P01160 compared with normal control group . Western blot analysis showed overexpression of P25103 protein level exited in P01160 group . Statistical analysis revealed correlation between P25103 mRNA and P05164 ( r=0.83 , P < 0.01 ) and LCP ( r=0.79 , P < 0.01 ) respectively . With immunohistochemistry staining , moderate to strong P25103 immunoreactivity was localized to alveolar membrane , I epithelium , II epithelium and polymorphonuclear leukocytes in the lung of P01160 . CONCLUSION : In P01160 , overexpression of P25103 contributes to disturbance of neuropeptides loop , resulting in aggregation of neutrophilic granulocyte and promoting deterioration of lung injury . Potentiator ivacaftor abrogates pharmacological correction of ΔF508 P13569 in cystic fibrosis . Cystic fibrosis ( CF ) is caused by mutations in the CF transmembrane conductance regulator ( P13569 ) . Newly developed " correctors " such as DB09280 ( VX-809 ) that improve P13569 maturation and trafficking and " potentiators " such as ivacaftor ( VX-770 ) that enhance channel activity may provide important advances in CF therapy . Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation ( G551D ) that affects only channel activity , a single compound is not sufficient to treat patients with the more common P13569 mutation , ΔF508 . Thus , patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit . However , whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro , the impact of chronic therapy has not been established . In studies of human primary airway epithelial cells , we found that both acute and chronic treatment with VX-770 improved P13569 function in cells with the G551D mutation , consistent with clinical studies . In contrast , chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated P13569 correction in ΔF508 homozygous cultures . This result reflected the destabilization of corrected ΔF508 P13569 by VX-770 , markedly increasing its turnover rate . Chronic VX-770 treatment also reduced mature wild-type P13569 levels and function . These findings demonstrate that chronic treatment with P13569 potentiators and correctors may have unexpected effects that can not be predicted from short-term studies . Combining these drugs to maximize rescue of ΔF508 P13569 may require changes in dosing and/or development of new potentiator compounds that do not interfere with P13569 stability . Growth factors expression in patients with erosive esophagitis . Although the pathogenesis and treatment of erosive esophagitis ( EE ) is well recognized , little is known about the cellular and molecular mechanisms of mucosal healing in EE patients . In this pilot study , we enrolled typical EE patients to evaluate what kinds of growth factors and their receptors were activated in their injured esophageal mucosa . Forty endoscopically proved EE patients were consecutively enrolled . Messenger RNA expressions , which includes keratinocyte growth factor ( KGF ) and its receptor ( P21802 ) , epidermal growth factor ( P01133 ) and its receptor ( P00533 ) , hepatocyte growth factor ( P14210 ) and its receptor ( HGFR ) , basic fibroblast growth factor ( P09038 ) , vascular endothelial growth factor ( P15692 ) , and cyclooxygenase ( P36551 ) -1 and P35354 , were measured using real-time polymerase chain reaction ( PCR ) . Data were compared between the injured EE mucosa and their normal esophageal mucosa above EE . The mRNA expressions of P14210 , HGFR , P01133 , P15692 , and P35354 , but not P00533 , KGF , P21802 , P09038 , and P23219 , were significantly increased in the injured mucosa of EE patients compared with those of normal mucosa ( P < 0.05 ) . The study found that P14210 , HGFR , P01133 , P15692 , and , P35354 are activated in the injured mucosa of EE patients ; their activation might be involved in mucosal repair and ulcer healing of EE . Rationalizing cyclooxygenase ( P36551 ) inhibition for maximal efficacy and minimal adverse events . New information indicates that cyclooxygenase-2 ( P35354 ) is constitutively expressed in several tissues , including brain , lung , pancreas , kidney , and ovary , and plays an important role in renal and gastrointestinal function . Selective P35354 inhibition has been associated in animal studies with impairment of ulcer healing and renal function and inhibition of prostacyclin , an effect that inhibits vasodilation without inhibiting platelet aggregation . The clinical consequences , if any , of these effects remain to be determined in long-term studies in humans . The premise that selective P35354 inhibitors will cause less gastrointestinal toxicity than nonsteroidal antiinflammatory drugs that inhibit both P36551 isoforms needs to take into account the low toxicity of nabumetone . The gastrointestinal safety profile of this nonacidic , dual P36551 inhibitor that does not undergo enterohepatic circulation has been evaluated in extensive clinical trials . The data submitted to the US Food and Drug Administration in the New Drug Application for nabumetone ( DB00461 ) , the comparative trials subsequently completed , the published databases of the comparative gastrointestinal toxicity of various nonsteroidal anti-inflammatory drugs ( NSAIDs ) , and the meta-analysis published in this issue of The American Journal of Medicine ( Schoenfeld , page 48S ) indicate that nabumetone has the lowest incidence of gastrointestinal toxicity among the extensively studied NSAIDs . Overall , the incidence is approximately 10-fold less than with comparator drugs . This rate is an appropriate current reference against which the gastrointestinal toxicity of P35354 inhibitors can be compared . Effects of pemetrexed , gefitinib , and their combination on human colorectal cancer cells . PURPOSE : The study investigated the effects of pemetrexed , gefitinib , and their combination on human colorectal cancer cells . METHODS : Six human colorectal cancer cells were exposed to pemetrexed , gefitinib , and their combination . Antitumor effects were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay . P04818 ( TS ) mRNA expression and P00533 mutation were studied by real-time RT-PCR and DNA sequence determination . Pharmacological interaction was studied using the combination index method . Cell cycle distribution and apoptosis were determined by flow cytometry . Activity assay was performed to assess the effects of drugs on TS activity , and Western blot was performed to assess the protein expression of pEGFR , pAKT , and pERK 1/2 . RESULTS : Six colorectal cancer cells are all sensitive to pemetrexed , and TS gene expression of cells was negatively related to pemetrexed sensitivity . The cytotoxic synergism was observed in concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib . The combination of pemetrexed and gefitinib modulated cell cycle and induced apoptosis . DB00642 combined with gefitinib decreased TS mRNA expression and in situ activity . DB00642 induced an P00533 -mediated activation of the phosphatidylinositol 3-kinase/AKT and P29323 pathway , which was inhibited by gefitinib . CONCLUSIONS : DB00642 is a promising agent , and pemetrexed combined with gefitinib has a significantly synergistic effect on colorectal cancer cells , which seems to present a strategy of pemetrexed combined with P00533 -TKIs in colorectal cancer treatment . DB03843 - or adjuvant-induced peripheral inflammation increases neurokinin-1 receptor gene expression in the mouse . Substance P ( SP ) has been widely studied as a mediator of nociception . The release of SP from primary afferent neurons is increased during nociception , and SP activates neurokinin-1 ( NK-1 ) receptors in the spinal cord and periphery . Nociception-evoked alterations in P25103 gene expression have been studied in rat models of persistent pain but have not been characterized in any murine models of peripheral inflammation . This study assessed behavioral responses and P25103 mRNA gene expression in mice receiving formalin or Freund 's complete adjuvant ( O75347 ) as an inflammatory stimulus . Mechanical withdrawal thresholds were measured before injection of formalin or O75347 and hind paw licking/biting timed during the late-phase of the formalin response . Two and 24 hours after formalin or O75347 injection , mechanical withdrawal thresholds were measured and the mice euthanized . Solution hybridization-nuclease protection assays were used to quantify P25103 mRNA levels . Results demonstrated that inflamed hind paws were edematous , and the withdrawal thresholds of the inflamed hind paws were significantly lower after formalin or O75347 injection . Neurokinin-1 receptor mRNA levels in the ipsilateral dorsal spinal cords of mice were higher at 24 h after formalin injection or 4 days after O75347 injection . These results confirm that mice are hyperalgesic at late time points after formalin or adjuvant injection when P25103 gene expression is elevated in the dorsal spinal cord . This supports the hypothesis that increased P25103 gene expression contributes to the development and maintenance of a hyperalgesic state . Ductal adenocarcinoma of the pancreas usually retained Q13485 and p53 protein status as well as expression of epithelial-to-mesenchymal transition markers and cell cycle regulators at the stage of liver metastasis . There are limited data on the biology of metastatic pancreatic ductal adenocarcinoma ( PDAC ) . The aim of the present study was to compare the expression of immunohistochemical markers that may be involved in the development of metastatic disease in primary PDAC and in synchronous liver metastatic tissues . Thirty-two stains ( corresponding to proteins encoded by 31 genes : Q13485 , P04637 , P62736 , CDH1 , P38936 , O95832 , O14493 , O95471 , P35222 , P00533 , P04626 , P02751 , P08727 , P28482 / P27361 , Q16539 , P46013 , P08253 , P14780 , P15941 ( 3 antibodies ) , P98088 , Q6W4X9 , P42345 , MYC , P48681 , P35354 , P62753 , P23443 , P01137 , P36897 , P08670 ) were evaluated using tissue microarray of 26 pairs of primary PDACs and their liver metastases . There were no significant differences in expression levels of examined proteins between primary and secondary lesions . In particular , metastatic PDAC retained the primary tumour 's Q13485 protein status in all and p53 protein status in all but one case . This surprising homogeneity also involved expression levels of markers of epithelial-to-mesenchymal transition as well as cell cycle regulators studied . In conclusion , the biological profiles of primary PDACs and their liver metastases seemed to be similar . Molecular alterations of PDAC related to a set of immunohistochemical markers examined in the present study were already present at the stage of localized disease . Copy number analysis of 24 oncogenes : O15151 identified as a putative marker for low recurrence risk in non muscle invasive bladder cancer . Patients with non-muscle invasive bladder cancer ( NMIBC ) generally have a high risk of relapsing locally after primary tumor resection . The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease . We studied the copy number variations ( CNVs ) of 24 oncogenes ( O15151 , P04198 , Q9UM73 , P16234 , P10721 , P35968 , P00374 , P00533 , MET , SMO , P11362 , MYC , P00519 , P07949 , P24385 , P30279 , P11802 , Q00987 , Q96GD4 , P04626 , P11388 , O14965 , AR and P15056 ) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence . DB03843 -fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder ( TURB ) were used ; 23 patients had relapsed and 20 were disease-free after 5 years . Amplification frequencies were analyzed for all genes and O15151 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones ( 0.65 vs. 0.3 ; Fisher 's test p=0.023 ) . Recurrence-free survival analysis confirmed the predictive role of O15151 ( log-rank test p=0.041 ) . Our preliminary results indicate a putative role for the O15151 gene in predicting local recurrence of bladder cancer . Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients . [ Studies on antigenspecific immunosuppression : Influence of 6-mercaptopurine-BGG-conjugates on BGG binding cells ( author 's transl ) ] . The rosette forming cell ( P41440 ) response of different lymphoid organs ( spleen , lymph node , peripheral blood ) of guinea pigs was examined by immunocytoadherence . The method has been adapted to work with the soluble antigen BGG . The number of antigen binding cells following immunization with BGG in O75347 reached the highest level on day 6 in Ln. popliteus and on the 10th day in spleen and peripheral blood . It is shown that the highest level of antigen binding cells appears generally before the maximum of circulating antibodies . After i.p.-immunization to alum-adsorbed BGG we only detected a small number of RFCs . After pretreatment with 6-mercaptopurineI-25-BGG ( MP-BGG ) and a control substance ( toluylI-25-BGG ) over 8 days and in increasing doses we obtained an antigenspecific suppression of BGG binding cells following MP-BGG treatment . In contrast to these findings the antibody titres were only slightly decreased in the beginning of the immune response . Spline-fitting with a genetic algorithm : a method for developing classification structure-activity relationships . Classification methods allow for the development of structure-activity relationship models when the target property is categorical rather than continuous . We describe a classification method which fits descriptor splines to activities , with descriptors selected using a genetic algorithm . This method , which we identify as SFGA , is compared to the well-established techniques of recursive partitioning ( RP ) and soft independent modeling by class analogy ( SIMCA ) using five series of compounds : cyclooxygenase-2 ( P35354 ) inhibitors , benzodiazepine receptor ( BZR ) ligands , estrogen receptor ( ER ) ligands , dihydrofolate reductase ( P00374 ) inhibitors , and monoamine oxidase ( MAO ) inhibitors . Only 1-D and 2-D descriptors were used . Approximately 40 % of compounds in each series were assigned to a test set , " cherry-picked " from the complete set such that they lie outside the training set as much as possible . SFGA produced models that were more predictive for all but the P00374 set , for which SIMCA was most predictive . RP gave the least predictive models for all but the MAO set . A similar trend was observed when using training and test sets to which compounds were randomly assigned and when gradually eliminating compounds from the ( designed ) training set . The stability of models was examined for the random and reduced sets , where stability means that classification statistics and the selected descriptors are similar for models derived from different sets . Here , SIMCA produced the most stable models , followed by SFGA and RP . We show that a consensus approach that combines all three methods outperforms the single best model for all data sets . Human mitochondrial import receptor , Tom20p . Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins . The cytosolic domain of the human mitochondrial protein import receptor , hTom20 , has been expressed as a fusion protein with glutathione S-transferase ( Q86UG4 ) in bacteria and the purified protein immobilized on Sepharose beads . To discriminate between specific binding of precursor proteins with the receptor and non-specific binding , precursors were recovered as a complex with Q86UG4 -hTom20 following competitive elution from the beads with reduced glutathione . Here , we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom20 interacts directly with the transcription-translation product of precursor proteins that bear a diverse array of targeting signals . Such proteins include a matrix protein ( pODHFR ) , a polytopic integral protein of the inner membrane ( uncoupling protein ) , a beta-barrel protein of the outer membrane ( P21796 /porin ) as well as bitopic integral proteins which are inserted into the outer membrane by either an NH2-terminal or COOH-terminal signal anchor sequence ( yTom70(1-29) P00374 and Bcl-2 , respectively ) . Application of HapMap data to the evaluation of 8 candidate genes for pediatric slow transit constipation . BACKGROUND : Slow transit constipation ( P52823 ) affects up to 3 % of all children and is an increasingly recognized cause of chronic constipation in children . We conducted a pilot study to investigate whether genes encoding neurotransmitters ( P20366 , Q9UHF0 , P01282 , NOS1 ) and receptors ( P25103 , P21452 , P29371 , P10721 ) could be responsible for P52823 . METHODS : One hundred seventeen tag single nucleotide polymorphisms ( SNPs ) , distributed among the candidate genes , were selected from HapMap data and genotyped using Sequenom ( San Diego , CA ) technology in 35 affected families . Evaluation of association was performed by transmission disequilibrium test and multilocus analysis . RESULTS : Five SNPs ( rs3771863 , rs4580655 , rs11722288 , rs4563545 , and rs3782221 ) in the P25103 , P29371 , P10721 , and NOS1 genes were found to be potentially associated with P52823 , although the significance of these results does not withstand correction for multiple testing . CONCLUSIONS : Our data indicate that 5 SNPs in the NOS1 , P25103 , P29371 , and P10721 genes could be involved in P52823 , especially rs3771863 in intron 1 of P25103 , which showed the highest association . New data integrating multitargeted antifolates into treatment of first-line and relapsed non-small-cell lung cancer . Non-small-cell lung cancer ( NSCLC ) represents approximately 80 % of all lung cancers . With modern platinum- based combination regimens , overall median survival has reached 9-12 months . Antifolates are active against several solid tumors and hematologic malignancies . The cytotoxic action of antifolates is mainly related to their ability to inhibit several different folate-dependent enzymes involved in DNA synthesis . DB00642 is a novel multitargeted antifolate that inhibits at least 3 of the enzymes involved in purine and pyrimidine synthesis : thymidylate synthase ( TS ) , dihydrofolate reductase ( P00374 ) , and glycinamide ribonucleotide formyltransferase ( GARFT ) . DB00642 was approved for the treatment of relapsed NSCLC as it produced equivalent response and survival rates and less toxicity compared with docetaxel . DB00642 in combination with platinum analogues or with gemcitabine showed equivalent clinical impact compared with standard combinations of platinum plus third-generation agents . We analyze the potential implications of pemetrexed 's role in first-line chemotherapy of NSCLC as well as hints of differential cytotoxic action according to histology , new schedules of vitamin supplementation , and target enzymes expression levels . Issues of pharmacogenomics are becoming relevant in defining pemetrexed efficacy . Chemosensitivity was significantly linked to low levels of TS , GARFT , and P00374 in preclinical models . Consequently , the differential expression of TS according to histology might explain the different activity of pemetrexed according to histology , as recently postulated . Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents : Part II . Antifolates that inhibit the key enzymes thymidylate synthase ( TS ) and dihydrofolate reductase ( P00374 ) have found clinical utility as antitumor and antiopportunistic agents . DB00563 { MTX , ( 1 ) } and 5-fluorouracil ( DB00544 ) were among the first clinically useful P00374 and TS inhibitors , respectively . The development of resistance to DB00544 , its occasional unpredictable activity and toxicity resulted in the search of novel antifolates . DB00642 ( 4 ) and raltitrexed ( 5 ) are newer antifolates that specifically inhibit TS , and are clinically useful as antitumor agents . A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase ( Q05932 ) . Recently , classical antifolates that do not need to be polyglutamylated have also been developed and include plevitrexed ( 6 ) and GW1843 ( 7 ) . Nolatrexed ( 8 ) , trimethoprim { P54849 , ( 11 ) } and piritrexim { PTX , ( 12 ) } are nonclassical antifolates for antitumor and parasitic chemotherapy that passively diffuse into cells and hence do not have to depend on Q05932 or the reduced folate carrier ( P41440 ) . Structural requirements for inhibition with antifolates have been studied extensively and novel agents that exploit key interactions in the active site of TS , P00374 , Q05932 , and P41440 have been proposed . This two-part review discusses the design , synthesis and structural requirements for TS and P00374 inhibition and their relevance to antitumor and parasitic chemotherapy , since 1996 . Monocyclic and 6-5 fused bicyclic antifolates were discussed in Part I . The 6-6 bicyclic and tricyclic antifolates will be discussed here in Part II .