MH_dev_71

Managing the underlying cause of cystic fibrosis : a future role for potentiators and correctors . Cystic fibrosis ( CF ) , a severe genetic disease , is caused by mutations that alter the structure and function of P13569 REA , a plasma membrane channel permeable to chloride and bicarbonate . Defective anion transport in CF irreversibly damages the lungs , pancreas , liver , and other organs . CF mutations cause loss of P13569 REA function in multiple ways . In particular , class 3 mutations such as p . Gly 551Asp strongly decrease the time spent by P13569 REA in the open state ( gating defect ) . Instead , class 2 mutations impair the maturation of P13569 REA protein and its transport from the endoplasmic reticulum to the plasma membrane ( trafficking defect ) . The deletion of phenylalanine 508 ( p . Phe 508del ) , the most frequent mutation among CF patients ( 70-90 % ) , destabilizes the P13569 REA protein , thus causing both a trafficking and a gating defect . These two defects can be overcome with drug-like molecules generically called correctors and potentiators , respectively . The potentiator Kalydeco ™ ( also known as DB08820 MEN or VX - 770 ) , developed by Vertex Pharmaceuticals , has been recently approved by the US FDA and the European Medicines Agency ( P15941 REA ) for the treatment of CF patients carrying at least one P13569 REA allele with the p . Gly 551Asp mutation ( 2-5 % of all patients ) . In contrast , the corrector VX - 809 , which significantly improves p . Phe 508del - P13569 REA trafficking in vitro , is still under study in clinical trials . Because of multiple defects caused by the p . Phe 508del mutation , it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action . This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect .

A phase II study of pralatrexate with vitamin B12 and folic acid supplementation for previously treated recurrent and / or metastatic head and neck squamous cell cancer . BACKGROUND : DB06813 SUB ( Fotolyn ( TM ) ; Allos Therapeutics Inc . ) is an antifolate dihydrofolate reductase ( P00374 REA ) inhibitor . We conducted a phase II study of pralatrexate with folic acid and B12 supplementation in patients with recurrent and / or metastatic head and neck squamous cell cancer ( R / M HNSCC ) . PATIENTS AND METHODS : This was a single-arm , Simon optimal two stage phase II study . Patients with R / M HNSCC previously treated with chemotherapy were eligible . The study was initiated with a dosing schedule of pralatrexate 190 mg / m ( 2 ) biweekly on a 4 - week cycle with vitamin supplementation . Due to toxicity concerns , the dosing was modified to 30 mg / m ( 2 ) weekly for 3 weeks in a 4 - week cycle with vitamin supplementation . Radiologic imaging was to be obtained about every 2 cycles . RESULTS : Thirteen subjects were enrolled ; 12 were treated . Seven of the twelve patients had previously received ≥ 2 lines of chemotherapy . The most common grade 3 toxicity was mucositis ( 3 patients ) . Seven patients did not complete two cycles of therapy due to progression of disease ( 4 ) , toxicity ( 1 ) , death ( 1 ) , and withdrawal of consent ( 1 ) . Two deaths occurred : one due to disease progression and the other was an unwitnessed event that was possibly related to pralatrexate . No clinical activity was observed . The median overall survival was 3.1 months . The study was closed early due to lack of efficacy . CONCLUSIONS : DB06813 SUB does not possess clinical activity against previously treated R / M HNSCC . Evaluation of pralatrexate in other clinical settings of HNSCC management with special considerations for drug toxicity may be warranted .

Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via DB02527 and P00533 REA - Src-MEK pathways . In autosomal-dominant polycystic kidney disease ( ADPKD ) , renal cysts develop by aberrant epithelial cell proliferation and transepithelial fluid secretion . We previously showed that ouabain increases proliferation of cultured human ADPKD cells via stimulation of the P01133 REA receptor ( P00533 REA ) - Src-MEK / P29323 REA signaling pathway . We examined whether ouabain affects fluid secretion and in vitro cyst growth of human ADPKD cell monolayers , ADPKD cell microcysts cultured in a three-dimensional collagen matrix , and metanephric organ cultures from Pkd 1 ( m1Bei ) mice . Physiological concentrations of ouabain alone did not affect net transepithelial basal-to-apical fluid transport in ADPKD monolayers or growth of cultured ADPKD microcysts . In contrast , in the presence of forskolin or 8 - bromo - DB02527 , ouabain significantly enhanced ADPKD fluid secretion and microcyst expansion . Ouabain exerted this effect by enhancing DB02527 - dependent Cl ( - ) secretion via the P13569 REA . Similarly , ouabain accelerated DB02527 - dependent cyst enlargement in Pkd 1 ( m1Bei ) mice metanephroi , with a more prominent response in homozygous than heterozygous mice . Ouabain had no effect on fluid secretion and cystogenesis of normal human kidney cells and caused only slight cystic dilations in wild-type mouse kidneys . The effects of ouabain in ADPKD cells and Pkd 1 ( m1Bei ) metanephroi were prevented by inhibitors of P00533 REA ( AG1478 ) , Src ( Q99463 ) , and MEK ( U0126 ) . Together , our results show that ouabain , used in physiological concentrations , has synergistic effects on DB02527 - mediated fluid secretion and cyst growth , via activation of the P00533 REA - Src-MEK pathway . These data provide important evidence for the role of ouabain as an endogenous hormone that exacerbates ADPKD cyst progression .

LC-MS based assay to measure intracellular compound levels in Mycobacterium smegmatis : linking compound levels to cellular potency . P00374 REA ( P00374 REA ) plays a central role in maintaining cellular pool of tetrahydrofolic acid , a cofactor necessary for DNA , RNA and protein synthesis . The clinical validation of P00374 REA as antibacterial target was established by the success of trimethoprim ( P54849 ) . P00374 REA is also an attractive target for identifying anti-tuberculosis molecules however , due to observed weak cellular potency , no P00374 REA inhibitors have been developed as drugs so far . P54849 and its analogs have poor cellular potency on Mycobacterium tuberculosis and Mycobacterium smegmatis cells . We found a mutant strain of M . smegmatis , mc² 155 to be sensitive to P54849 whereas wild type strain was not inhibited by P54849 . We utilized this system to probe if poor or lack of activity of P54849 is a consequence of poor intracellular compound levels . An LC-MS based method was developed for measuring P54849 and rifampicin ( Q9HBH0 ) in M . smegmatis . Using the assay , equivalent Q9HBH0 levels were observed in both strains however , P54849 was detected only in mc² 155 cells , hence proving a positive correlation between potency and compound levels . To the best of our knowledge this is the first time LC-MS method has been used to measure compound levels in mycobacterial cells . We propose it to be a valuable tool to understand the lack of potency or resistance mechanisms in antimycobacterial drug development .

Augmentation by citalopram of risperidone-induced monoamine release in rat prefrontal cortex . RATIONALE : A typical antipsychotics ( APDs ) , e . g . olanzapine and risperidone , have been reported to be effective adjunctive treatment for depression if selective serotonin ( 5 - HT ) reuptake inhibitors ( SSRIs ) alone are ineffective . OBJECTIVES AND METHODS : We utilized microdialysis in awake , freely moving rats to study the effect of risperidone in combination with citalopram , an SSRI , on extracellular 5 - HT , dopamine ( DA ) , and norepinephrine ( NE ) efflux in rat medial prefrontal cortex ( mPFC ) . RESULTS : DB00734 MEN ( 1.0 mg / kg , s . c . ) , given alone , significantly increased 5 - HT , DA , and NE concentrations in the mPFC . DB00215 ( 10 mg / kg , s . c . ) , by itself , produced a significant increase in 5 - HT levels only . The combination of risperidone and citalopram produced significantly greater increases in efflux of both DA and NE than risperidone alone . However , the effect of this combination on extracellular 5 - HT concentrations was not significantly different than that of citalopram alone . The augmentation of DA and NE efflux induced by risperidone plus citalopram could be partially blocked by the selective P08908 REA antagonist , WAY 100635 ( 0.2 mg / kg , s . c . ) . CONCLUSIONS : The results suggest that the ability of atypical APDs to augment the therapeutic efficacy of SSRIs in major depression and treatment-resistant depression may be due , at least in part , to potentiation of SSRI-induced increases in cortical DA and NE . The contributions of P08908 REA receptor stimulation and 5 - Q13049 REA and alpha 2 adrenergic receptor antagonism to this augmentation are discussed .

Gating properties of Q14524 REA mutations and the response to mexiletine in long-QT syndrome type 3 patients . BACKGROUND : DB00379 MEN ( Mex ) has been proposed as a gene-specific therapy for patients with long-QT syndrome type 3 ( LQT 3 ) caused by mutations in the cardiac sodium channel gene ( Q14524 REA ) . The degree of QT shortening and the protection from arrhythmias vary among patients harboring different mutations . We tested whether the clinical response to Mex in LQT 3 could be predicted by the biophysical properties of the different mutations . METHODS AND RESULTS : We identified 4 Q14524 REA mutations in 5 symptomatic LQT 3 patients with different responses to Mex ( 6 to 8 mg . kg ( - 1 ) . d ( - 1 ) ) . We classified the mutations as sensitive to Mex ( P1332L , R1626P ; > /= 10 % of QTc shortening and QTc < 500 ms or no arrhythmias ) or insensitive to Mex ( S941N , M1652R ; negligible or no QTc shortening and sudden death ) . We measured Na ( + ) current from P29320 REA 293 cells transfected with wild-type ( WT ) or mutant Nav 1.5 . All mutations showed impaired inactivation of Na ( + ) current , but the mutations identified in patient responders to Mex ( P1332L , R1626P ) showed a hyperpolarizing shift of V ( 1/2 ) of steady-state inactivation . Furthermore , Mex produced use-dependent block with the order R1626P =P 1332L > S941N = WT > M1652R , suggesting that Mex-sensitive mutants present prolonged recovery from Mex block . CONCLUSIONS : We propose that voltage dependence of channel availability and shifts of V ( 1/2 ) of steady-state inactivation correlate with the clinical response observed in LQT 3 patients . This supports the view that the response to Mex is mutation specific and that in vitro testing may help to predict the response to therapy in LQT 3 .

Celecoxib with chemotherapy in colorectal cancer . P35354 REA ( P35354 REA ) is the enzyme that normally synthesizes prostaglandins during an inflammatory response . Many primary and metastatic cancers express P35354 REA , and its presence is correlated with tumor angiogenesis , more invasive tumor phenotype , resistance to apoptosis , and systemic immunosuppression . The expression of P35354 REA is associated with a worse prognosis . Inhibition of prostaglandin synthesis may be beneficial in human malignancy . Regular consumption of nonsteroidal anti-inflammatory drugs ( NSAIDs ) decreases the incidence of , and mortality rate resulting from , a number of types of gastrointestinal cancers . Premalignant colonic lesions regress following the administration of nonspecific P36551 REA inhibitors , such as sulindac ( DB00605 MENMAX DB00605 MEN ) . Advanced solid tumor patients treated with indomethacin ( DB00328 ) survive twice as long as do such patients who receive supportive care alone . The U . S . Food and Drug Administration has approved specific P35354 REA inhibitors for the treatment of arthritis , pain , and familial adenomatous polyposis . Preclinical studies show that these drugs block angiogenesis , suppress solid tumor metastases , and slow the growth of implanted gastrointestinal cancer cell lines . The P35354 REA inhibitors have safely and effectively been combined with chemotherapeutic agents in experimental studies . Ongoing clinical trials are currently assessing the potential therapeutic role of P35354 REA inhibitors in both prevention and treatment of a diverse range of human cancers .

Characterization of the aggregation responses of camel platelets . BACKGROUND : Despite evidence of active hemostasis , camel platelets barely respond to common aggregating agents at standard doses used for human platelet aggregation . OBJECTIVES : The purpose of the study was to find out whether camel platelets can be activated by high doses or combinations of aggregation agonists , and to characterize the receptor that mediates the aggregation response to adenosine diphosphate ( ADP ) , the most potent agonist for camel platelets known so far . METHODS : Aggregation studies were performed with platelet-rich plasma ( PRP ) in response to multiple doses or combinations of ADP , epinephrine ( P08473 REA ) , collagen , and arachidonic acid ( AA ) . Aggregation responses to ADP were performed before and after the addition of the ADP receptor ( Q9H244 REA ) antagonist DB00758 MEN . RESULTS : Camel platelets responded to ADP at doses higher than the standard dose for human platelets , and to combinations of P08473 REA and other agonists , while no aggregation was elicited with P08473 REA or AA alone . DB00758 MEN blocked the ADP-induced aggregation responses in a dose-dependent fashion in vitro . CONCLUSIONS : Camel platelet aggregation can be activated by increasing the dose of some agonists such as ADP , but not AA or P08473 REA . Irreversible aggregation of camel platelets could also be triggered by a combination of P08473 REA and ADP , and collagen and AA . Inhibition with clopidogrel suggests that camel platelets express the ADP receptor , Q9H244 REA . Understanding platelet function in camels will add to the understanding of platelet function in health and disease .

Extracellular signal-regulated kinase / mitogen-activated protein kinases block internalization of delta-opioid receptors . Translocation of G protein-coupled receptors ( GPCRs ) from the cell membrane to cytosol depends on the kind of ligand activating the receptor . This principle is clearly demonstrated for opioid receptors , because diverse opiate agonists rapidly induce receptor internalization , whereas morphine almost fails . We report here the impact of mitogen-activated protein ( Q96HU1 ) kinase isoforms extracellular signal-regulated kinase ( P29323 REA ) 1/2 on the internalization of delta-opioid receptors ( DORs ) expressed in human embryonic kidney ( P29320 REA ) 293 cells . Receptor activation by etorphine turned out to transiently phosphorylate P29323 REA / Q96HU1 kinases and bring about Q8IXH6 internalization within 20 min . In contrast , prolonged exposure of HEK 293 cells to morphine excited persistent phosphorylation of P29323 REA / Q96HU1 kinases , and those cells failed to internalize the opioid receptor . When P29323 REA / Q96HU1 kinase phosphorylation was blocked by 2 ' - Amino - 3 ' - methoxyflavone ( PD98059 ) , morphine gained the ability to strongly induce Q8IXH6 endocytosis . The importance of activated Q96HU1 kinases for Q8IXH6 internalization is further demonstrated by glutamate and paclitaxel because these substances induce phosphorylation of P27361 REA / 2 and concomitantly prevent Q8IXH6 sequestration by etorphine . In addition , receptor internalization by morphine was facilitated by inhibition of protein kinase C and opioid-mediated transactivation of epidermal growth factor receptor ( P00533 REA ) , both activating P29323 REA / Q96HU1 kinases by opioids . The mechanism affording Q8IXH6 internalization by PD98059 may relate to arrestin , which uncouples GPCRs and thus triggers receptor internalization . Arrestin considerably translocates toward the cell membrane upon Q8IXH6 activation by morphine in presence of the Q96HU1 kinase blocker , but it fails in the absence of PD98059 . We conclude that P29323 REA / Q96HU1 kinase activity prevents opioid receptor desensitization and sequestration by blocking arrestin 2 interaction with activated DORs .

Flow cytometric analysis of mammalian glial cultures treated with methotrexate . DB00563 MEN ( MTX ) is an antineoplastic drug that acts by competitive inhibition of the enzyme dihydrofolate reductase ( P00374 REA ) . MTX treatment of cultured cell lines leads to the emergence of resistant cell populations . Studies using stepwise selection procedures have demonstrated that MTX resistance conferred by overproduction of P00374 REA can be caused by P00374 REA gene amplification . We examined the effect of MTX on cells whose origin more closely approximates the in vivo condition by developing a culture system using dissociated brain tissue from 17-19 day old mouse embryos . At the first passage , cultures were divided into control and MTX groups . Cells were treated with the same or successively higher concentrations of MTX at each passage over a 3-4 month period . The first passage eliminated neurons and left a glial culture comprised of approximately 90 % astrocytes . We used the Fluorescence Activated Cell Sorter in conjunction with fluorescent dyes to measure P00374 REA content , DNA content , size , and viability of glial cells following MTX treatment . MTX-treated cells divided but grew more slowly and were larger than untreated cells . Stepwise selection in 30/60 / 90 nM or 60/120 nM MTX resulted in significant two - to threefold increases in fluorescence , and hence P00374 REA levels . Slot hybridizations assays demonstrated a threefold increase in P00374 REA gene copy number in the DNA from the 30/60 / 90 cultures . Thus , our findings were consistent with the results obtained from somatic cell lines , and lend support to the hypothesis that gene amplification may be a common mechanism for the acquisition of resistance in many types of cells . They also indicate that glial cells may be a specific target for cytotoxic effects of MTX on the central nervous system .

Evaluation of the pharmacokinetics , preclinical and clinical efficacy of pralatrexate for the treatment of T-cell lymphoma . INTRODUCTION : Peripheral T-cell lymphomas ( PTCLs ) are a heterogeneous group of T-cell neoplasms . Most patients with PTCL have a poor outcome with conventional therapies and are not cured without stem-cell transplantation . DB06813 SUB , a novel antifolate chemotherapeutic agent , was rationally designed to impede folate metabolism by inhibiting dihydrofolate reductase ( P00374 REA ) and to be more efficiently internalized into tumor cells . DB06813 SUB is the first drug that is FDA approved for patients with relapsed and refractory PTCL . AREAS COVERED : DB06813 SUB has been used as a single agent and in combination with other agents in clinical trials for non-Hodgkin ' s lymphoma and Hodgkin ' s disease as well as in solid tumors . This review will cover the development of pralatrexate , the pharmacokinetics of pralatrexate , preclinical findings with pralatrexate and clinical studies of pralatrexate in hematologic malignancies . EXPERT OPINION : DB06813 SUB has significant activity in vitro , and in early Phase I / II trials , responses were noted in patients with aggressive T-cell lymphomas . The DB06813 SUB in Patients with Relapsed or Refractory Peripheral T-Cell Lymphoma trial demonstrated the activity of pralatrexate across a spectrum of heavily pretreated patients with different aggressive T-cell lymphoma subtypes , and studies in cutaneous T-cell lymphoma have shown efficacy at different doses and schedules . The most frequent adverse events in these trials were mucositis , reversible thrombocytopenia and fatigue .

DB09280 - DB08820 MEN in Patients with Cystic Fibrosis Homozygous for Phe 508del P13569 REA .

Distinct mechanistic activity profile of pralatrexate in comparison to other antifolates in in vitro and in vivo models of human cancers . PURPOSE : This study evaluated mechanistic differences of pralatrexate , methotrexate , and pemetrexed . METHODS : Inhibition of dihydrofolate reductase ( P00374 REA ) was quantified using recombinant human P00374 REA . Cellular uptake and folylpolyglutamate synthetase ( Q05932 REA ) activity were determined using radiolabeled pralatrexate , methotrexate , and pemetrexed in NCI-H 460 non-small cell lung cancer ( NSCLC ) cells . The tumor growth inhibition ( TGI ) was assessed using MV522 and NCI-H 460 human NSCLC xenografts . RESULTS : Apparent K ( i ) values for P00374 REA inhibition were 45 , 26 , and > 200 nM for pralatrexate , methotrexate , and pemetrexed , respectively . A significantly greater percentage of radiolabeled pralatrexate entered the cells and was polyglutamylatated relative to methotrexate or pemetrexed . In vivo , pralatrexate showed superior anti-tumor activity in both NSCLC models , with more effective dose-dependent TGI in the more rapidly growing NCI-H 460 xenografts . CONCLUSIONS : DB06813 SUB demonstrated a distinct mechanistic and anti-tumor activity profile relative to methotrexate and pemetrexed . DB06813 SUB exhibited enhanced cellular uptake and increased polyglutamylation , which correlated with increased TGI in NSCLC xenograft models .

Ca2 + - calmodulin and janus kinase 2 are required for activation of sodium-proton exchange by the Gi-coupled 5 - hydroxytryptamine 1a receptor . The type 1 sodium-proton exchanger ( P19634 REA ) is expressed ubiquitously and regulates key cellular functions , including mitogenesis , cell volume , and intracellular pH . Despite its importance , the signaling pathways that regulate P19634 REA remain incompletely defined . In this work , we present evidence that stimulation of the 5 - hydroxytryptamine 1A ( P08908 REA ) receptor results in the formation of a signaling complex that includes activated O60674 REA ( Jak 2 ) , Ca2 + / calmodulin ( P62158 ) , and P19634 REA , and which involves tyrosine phosphorylation of P62158 . The signaling pathway also involves rapid agonist-induced association of P62158 and P19634 REA as assessed by coimmunoprecipitation studies and by bioluminescence resonance energy transfer studies in living cells . We propose that P19634 REA is activated through this pathway : P08908 REA receptor --> G ( i2 ) alpha and / or G ( i3 ) alpha --> Jak 2 activation --> tyrosine phosphorylation of P62158 --> increased binding of P62158 to P19634 REA --> induction of a conformational change in P19634 REA that unmasks an obscured proton-sensing and / or proton-transporting region of P19634 REA --> activation of P19634 REA . The G ( i / o ) - coupled P08908 REA receptor now joins a handful of Gq-coupled receptors and hypertonic shock as upstream activators of this emerging pathway . In the course of this work , we have presented clear evidence that P62158 can be activated through tyrosine phosphorylation in the absence of a significant role for elevated intracellular Ca2 + . We have also shown for the first time that the association of P62158 with P19634 REA in living cells is a dynamic process .

DB00472 MEN induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats . DB00472 MEN ( FLX ) is a drug commonly used as antidepressant . However , its effects on tumorigenesis remain controversial . Aiming to evaluate the effects of FLX treatment on early malignant changes , we analyzed serotonin ( 5 - HT ) metabolism and recognition , aberrant crypt foci ( Q9NQ94 ) , proliferative process , microvessels , vascular endothelial growth factor ( P15692 REA ) , and cyclooxygenase - 2 ( P35354 REA ) expression in colon tissue . Male Wistar rats received a daily FLX-gavage ( 30mgkg ( - 1 ) ) and , a single dose of 1,2 dimethylhydrazine ( Q03001 REA ; i . p . , 125mgkg ( - 1 ) ) . After 6 weeks of FLX-treatment , our results revealed that FLX and nor-fluoxetine ( N-FLX ) are present in colon tissue , which was related to significant increase in serotonin ( 5 - HT ) levels ( P < 0.05 ) possibly through a blockade in P31645 REA mRNA ( serotonin reuptake transporter ; P < 0.05 ) resulting in lower 5 - hydroxyindoleacetic acid ( 5 - HIAA ) levels ( P < 0.01 ) and , P28335 REA receptor mRNA expressions . FLX-treatment decreased dysplastic Q9NQ94 development ( P < 0.01 ) and proliferative process ( P < 0.001 ) in epithelia . We observed a significant decrease in the development of malignant microvessels ( P < 0.05 ) , P15692 REA ( P < 0.001 ) , and P35354 REA expression ( P < 0.01 ) . These findings suggest that FLX may have oncostatic effects on carcinogenic colon tissue , probably due to its modulatory activity on 5 - HT metabolism and / or its ability to reduce colonic malignant events .

Purine receptor Q15077 REA mediates cellular response to γ-ray-induced DNA damage . We previously showed that nucleotide P2 receptor agonists such as DB00171 and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant P16104 REA ( γ P16104 REA ) , which is considered to be an indicator of DNA damage so far , by activating purine Q15077 REA and Q9H244 REA receptors . Therefore , we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage . In the present study , we tested this idea by using human lung cancer A549 cells . First , reverse-transcription polymerase chain reaction ( RT-PCR ) showed that Q15077 REA receptor is highly expressed in A549 cells , but Q9H244 REA receptor is only weakly expressed . Next , colony formation assay revealed that Q15077 REA receptor antagonist MRS 2578 markedly reduced the survival rate of γ-ray-exposed A549 cells . The survival rate was also significantly reduced in Q15077 REA - knock-down cells , compared with scramble siRNA-transfected cells . Since it has reported that phosphorylation of P27361 REA / 2 after activation of P00533 REA via Q15077 REA and Q9H244 REA receptors is involved in the repair response to γ-ray-induced DNA damage , we next examined whether γ-ray-induced phosphorylation of P27361 REA / 2 was also inhibited by MRS 2578 in A549 cells . We found that it was . Taken together , these findings indicate that purinergic signaling through Q15077 REA receptor , followed by P27361 REA / 2 activation , promotes the cellular repair response to γ-ray-induced DNA damage .

Purinergic receptors contribute to early mesangial cell transformation and renal vessel hypertrophy during angiotensin II-induced hypertension . Chronic P03950 REA II infusions lead to increases in intrarenal P03950 REA II levels , hypertension , and tissue injury . Increased blood pressure also elicits increases in renal interstitial fluid ( Q9HBH0 ) DB00171 concentrations that stimulate cell proliferation . We evaluated the contribution of purinergic receptor activation to P03950 REA II-induced renal injury in rats by treating with clopidogrel , a Q9H244 REA receptor blocker , or with PPADS , a nonselective P2 receptor blocker . alpha-Actin expression in mesangial cells , afferent arteriolar wall thickness ( AAWT ) , cortical cell proliferation , and macrophage infiltration were used as early markers of renal injury . DB00758 MEN and PPADS did not alter blood pressure , renin or kidney P03950 REA II content . alpha-Actin expression increased from control of 0.6 + / - 0.4 % of mesangial area to 6.3 + / - 1.9 % in P03950 REA II-infused rats and this response was prevented by clopidogrel ( 0.4 + / - 0.2 % ) and PPADS . The increase in AAWT from 4.7 + / - 0.1 to 6.0 + / - 0.1 mm in P03950 REA II rats was also prevented by clopidogrel ( 4.8 + / - 0.1 mm ) and PPADS . P03950 REA II infusion led to interstitial macrophage infiltration ( 105 + / - 16 vs . 62 + / - 4 cell / mm ( 2 ) ) and tubular proliferation ( 71 + / - 15 vs . 20 + / - 4 cell / mm ( 2 ) ) and these effects were prevented by clopidogrel ( 52 + / - 4 and 36 + / - 3 cell / mm ( 2 ) ) and PPADS . Q9HBH0 DB00171 levels were higher in P03950 REA II-infused rats than in control rats ( 11.8 + / - 1.9 vs . 5.6 + / - 0.6 nmol / l , P < 0.05 ) . The results suggest that activation of vascular and glomerular purinergic P2 receptors may contribute to the mesangial cell transformation , renal inflammation , and vascular hypertrophy observed in P03950 REA II-dependent hypertension .

Establishment of pemetrexed-resistant non-small cell lung cancer cell lines . DB00642 ( P15941 REA ) , a multitargeted antifolate with manageable toxicity , is active against non-squamous non-small cell lung cancer ; however , most patients eventually acquire resistance to P15941 REA . To elucidate the resistant mechanism , we established P15941 REA - resistant lung adenocarcinoma cell lines . Two parental cell lines , PC - 9 and A549 , were treated with step-wise increasing concentrations of P15941 REA . 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 REA ) , 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 REA ( 2.2- , 2.9- , 8.4- , and 14.3- fold , respectively ) . The four A549 sublines also showed more resistance to P15941 REA ( 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 REA - resistant sublines expressed more TS than the parental cells , by polymerase chain reaction and Western blotting . P00374 REA was significantly increased in the four P15941 REA - resistant A549 sublines . GARFT did not correlate with resistance to P15941 REA . In summary , P15941 REA - resistant cells remained sensitive to docetaxel , vinorelbine , 5 - fluorouracil , and irinotecan . TS expression appeared to be associated with resistance to P15941 REA .

[ Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis ] . DB01373 - sensitive forms of adenylyl cyclase ( AC ) were revealed in most vertebrates and invertebrates and also in some unicellular organisms , in particular ciliates . We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis . These cations at the concentrations of 0.2- 20 microM stimulated the enzyme activity , and maximum of catalytic effect was observed at 2 microM Ca2 + . DB01373 cations at a concentrations of 100 microM or higher inhibited the AC activity . P62158 antagonists W - 5 and W - 7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2 + and blocked the effect at higher concentrations of Ca2 + . DB00477 MEN , another calmodulin antagonist , reduced Ca2 + - stimulated AC activity only at the concentrations of 200-1000 microM . AC stimulating effects of serotonin , P01133 REA and DB02527 increased in the presence of 5 microM Ca2 + . AC stimulating effects of P01133 REA , DB02527 and insulin decreased in the presence of 100 microM Ca2 + , and AC stimulating effect of DB02527 decreased also in the presence of calmodulin antagonists ( 1 mM ) . At the same time , stimulating effect of D-glucose in the presence of Ca2 + and calmodulin antagonists did not change essentially . The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T . pyriformis which mediate enzyme stimulation by P01133 REA , DB02527 , insulin , and serotonin .

Suppression of tumor growth and metastasis by a P17948 REA antagonizing peptide identified from a phage display library . Although the P15692 REA - Flk - 1 - pathway has been known as the major driving force of angiogenesis , new evidence has shown that P17948 REA / Flt - 1 plays important roles during the neovascularization under pathological conditions including tumor , atherosclerosis and arthritis . In search of Flt - 1 receptor antagonizing peptides , we screened a phage display 12 - mer-peptide library with recombinant Flt - 1 protein . Seven candidate peptides were identified that specifically bound to P15692 REA receptor Flt - 1 , of which peptide F56 ( WHSDMEWWYLLG ) almost abolished P15692 REA binding to receptor Flt - 1 in vitro . In vivo , F56 fused with P00374 REA ( P00374 REA - F56 ) inhibited angiogenesis in a P62158 assay . Moreover , P00374 REA - F56 significantly inhibited the growth of nodules of human gastric cancer cell line MGC - 803 in BALB / c nude mice . Histological analyses showed that necrosis of the implanted tumor was markedly enhanced following treatment with P00374 REA - F56 . In the severe combined immunodeficiency disease ( SCID ) mouse model for studying metastasis of the human breast cancer cell line BICR-H 1 , synthetic peptide F56 significantly inhibited tumor growth and lung metastases . Taken together , our results have demonstrated that peptide F56 , as a Flt - 1 receptor antagonist , fulfilled the antiangiogenic and antimetastatic effects by specifically interfering with the interaction between P15692 REA and receptor Flt - 1 . Thus , short peptide F56 may have clinical potential in tumor therapy .

[ Effects of chronic fluoxetine treatment on catalepsy and immune response in mice genetically predisposed to freezing reaction : the role of P08908 REA and 5 - Q13049 REA receptors and tph 2 and P31645 REA genes ] . ASC / Icg ( Antidepressant Sensitive Catalepsy ) mouse strain selected for high predisposition to pinch-induced catalepsy is characterized by depressive-like behavior and impaired immune response . Chronic treatment with SSRI fluoxetine attenuated catalepsy manifestation and normalized a decreased number of rosette-forming cells ( P41440 REA ) in spleen in ASC mice . Chronic fluoxetine administration had no effect on catalepsy and P41440 REA number in mice of parental cataleptic CBA / Lac strain . DB00472 MEN failed to alter P08908 REA receptor functional activity in mice of both strains and diminished 5 - Q13049 REA receptor functional activity in CBA but not in ASC mice . No effect on cortical P08908 REA and 5 - Q13049 REA receptor mRNA levels and on P08908 REA receptor , tph 2 ( tryptophan hydroxylase - 2 ) and P31645 REA ( serotonin transporter ) mesencephalic gene expression was observed in ASC mice . Other possible serotonergic mechanisms of fluoxetine effect on catalepsy and immune response in mice with depressive-like state are discussed .

Order of genes on human chromosome 5q with respect to 5q interstitial deletions . Using ( a ) somatic cell hybrids retaining partial chromosome 5 and ( b ) clinical samples from patients with acquired deletions of the long arm of chromosome 5 , combined with chromosome 5 - linked DNA probes , some of which exhibited RFLPs , we have determined the order of a series of genes on chromosome 5 . The order established is 5pter - - - MLVI - 2 - - - cen - - - P07686 REA - - - P00374 REA - - - Pi227 - - - - cp12 . 6 - - - ( P05113 REA , P05112 REA ) - - - P08700 REA - - - P04141 REA - - - P05230 REA - - - ( P07333 REA , P09619 REA ) - - - ( treC , ADRBR ) - - - ( Q5SW96 - Q13585 , P09603 REA ) - - - qter . The suggested order and orientation for the closely linked P08700 REA / P04141 REA gene pair is cen - - - 5 ' P08700 REA 3 ' - - - 5 ' P04141 REA 3 ' - - - qter , on the basis of analysis of the P04141 REA rearrangement in HL60 DNA . The map position of the GRL locus , which was consistent with both somatic cell hybrid and 5q - analyses , was telomeric to P04141 REA and centromeric to P07333 REA / P09619 REA , near P05230 REA . Long-range restriction-enzyme analysis of 5q - DNAs did not detect rearrangements of 5q - linked probes except in HL60 DNA , but it did reveal putative long-range RFLPs of several loci . RFLPs for GRL , Pi227 , cp12 . 6 , P08700 REA , and P07333 REA can detect deletions in bone marrow and in leukemia cells from patients with acquired 5q deletions .

[ Presynaptic P08908 REA receptors in immunomodulation ] . It is shown that a selective agonist of P08908 REA receptors 8 - OH-DPAT in a low dose ( 0.1 mg / kg ) , which is known to affect mainly the presynaptic P08908 REA receptors increased the immune response at the peak of reactions ( the forth or fifth day after immunization with sheep red blood cells - SRBC ) in CBA mice and Wistar rats . Treatment of the animals with the drug 15 min prior to antigen injection raised the number of plaque-forming cells ( lgM - P27918 REA ) and rosette-forming cells ( P41440 REA ) in the spleen . The preliminary blockade of P08908 REA receptor with a selective antagonist of P08908 REA receptors WAY - 100635 ( 0.1 mg / kg ) prevented the immunostimulating effect of 5 - HT 1A receptors agonist 8 - OH-DPAT , whereas WAY - 100635 administration alone in the same dose did n't change the immune response . Activation of P08908 REA receptors under conditions of electrical lesion of 5 - HTergic neurons of the nucleus raphe was unable to enhance the immune reactions , as it did in sham-operated rats . The data obtained indicate that the somatodendric P08908 REA autoreceptors are involved in immunomodulation .

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 REA ) , epidermal growth factor ( P01133 REA ) and its receptor ( P00533 REA ) , hepatocyte growth factor ( P14210 REA ) and its receptor ( HGFR ) , basic fibroblast growth factor ( P09038 REA ) , vascular endothelial growth factor ( P15692 REA ) , and cyclooxygenase ( P36551 REA ) - 1 and P35354 REA , 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 REA , HGFR , P01133 REA , P15692 REA , and P35354 REA , but not P00533 REA , KGF , P21802 REA , P09038 REA , and P23219 REA , were significantly increased in the injured mucosa of EE patients compared with those of normal mucosa ( P < 0.05 ) . The study found that P14210 REA , HGFR , P01133 REA , P15692 REA , and , P35354 REA are activated in the injured mucosa of EE patients ; their activation might be involved in mucosal repair and ulcer healing of EE .

Glucocorticoid-induced surface expression of annexin 1 blocks beta 2 - integrin adhesion of human eosinophils to intercellular adhesion molecule 1 surrogate protein . BACKGROUND : Glucocorticoids attenuate the population of eosinophils and T lymphocytes in asthmatic airways . The decrease in airway eosinophilia is caused both by accelerated cell death and by induction of blockade of integrin adhesion . In this study , we examined the hypothesis that annexin 1 surface expression , which is upregulated by the glucocorticoid receptor , prevents integrin adhesion essential to cell migration by blocking intracellular translocation of cytosolic group IV phospholipase A2 ( P47712 REA ) . OBJECTIVE : To examine the relationship of the glucocorticoid on annexin 1 expression and the effect of blockade of annexin 1 activity on adhesion of human eosinophils in vitro . To determine the relationship between annexin 1surface expression and nuclear membrane translocation of P47712 REA . METHODS : Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate ( FP ) , and beta 2 - integrin adhesion was measured after stimulation with P05113 REA or eotaxin . Effects of FP on P47712 REA expression , phosphorylation , and translocation were determined . The role of annexin 1 was examined by using annexin 1 blocking antibody and / or mimetic peptides . RESULTS : DB00588 MEN decreased stimulated eosinophil adhesion and caused 4 - fold increase in annexin 1 expression on the plasma membrane . Inhibition of adhesion by FP was blocked with annexin 1 blocking antibody . Annexin 1 N-terminal mimetic peptide also blocked beta 2 - integrin adhesion . Translocation of P47712 REA to the nuclear membrane was significantly blocked by incubation with FP . Blockade was reversed with annexin 1 blocking antibody . CONCLUSION : Blockade of beta 2 - integrin adhesion by glucocorticoid is regulated by annexin 1 , which blocks P47712 REA translocation to nuclear membrane .

DB06813 SUB : a novel synthetic antifolate for relapsed or refractory peripheral T-cell lymphoma and other potential uses . PURPOSE : The pharmacology , pharmacokinetics , clinical trials , adverse effects , dosage , and economic considerations of pralatrexate ( DB06813 SUB ) are reviewed . SUMMARY : Peripheral T-cell lymphoma ( PTCL ) comprises approximately 15-20 % of all aggressive lymphomas and 5-10 % of all non-Hodgkin ' s lymphomas . Advanced PTCL is often refractory to traditional first-line chemotherapy regimens . DB06813 SUB was developed as a synthetic folate analog antimetabolite that competitively inhibits dihydrofolate reductase ( P00374 REA ) . This results in the depletion of thymidine , leading to interference with deoxyribonucleic acid synthesis and cancer cell death . DB06813 SUB has a higher potency than methotrexate and edatrexate ( EDX ) . The efficacy and safety of DB06813 SUB have been demonstrated in the PROPEL trial , a prospective phase II trial in patients with relapsed or refractory PTCL . Patients with prior stem cell transplantation receiving DB06813 SUB also had similar response rates ( RRs ) . DB06813 SUB was investigated on the treatment of relapsed or refractory cutaneous T-cell lymphoma , previously treated advanced non-small cell lung cancer and other solid malignancies . DB06813 SUB has similar side effects to other P00374 REA inhibitors . The most common side effect of DB06813 SUB is mucositis . The recommended dose of DB06813 SUB is 30 mg / m ( 2 ) weekly once for 6 weeks in 7 - week cycle until disease progresses or unacceptable toxicity for PTCL and may require dose reduction or discontinuation . Patients should be supplemented with oral folic acid and intramuscular vitamin B ( 12 ) injections . CONCLUSION : DB06813 SUB provides clinical benefit to patients with relapsed or refractory PTCL with durable complete and partial responses in patients who had not responded to multiple prior treatment regimens .

Development of stable cell lines expressing different subtypes of GABAA receptors . The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors . For this , we developed an original two step selection strategy , in which the first step consisted of transfecting P29320 REA 293 cells with rat GABAA receptor alpha and beta subunits . G 418 resistant colonies isolated at this step were screened for [ 3H ] muscimol binding to select for those that coexpressed alpha - and beta-subunits . The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant P00374 REA gene . After a second round of selection , this time in presence of methotrexate , those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [ 3H ] flumazenil as a probe . This strategy was applied to the isolation of 3 GABAA receptor clones , alpha 1 beta 2 gamma 2s , alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s , that expressed relatively high levels of these proteins . These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations . These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes .

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 REA ) inhibitors , benzodiazepine receptor ( BZR ) ligands , estrogen receptor ( ER ) ligands , dihydrofolate reductase ( P00374 REA ) 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 REA 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 .

Single agent and combination studies of pralatrexate and molecular correlates of sensitivity . BACKGROUND : DB06813 SUB is a dihydrofolate reductase ( P00374 REA ) inhibitor with high affinity for reduced folate carrier 1 ( P41440 REA - 1 ) and folylpolyglutamate synthetase ( Q05932 REA ) , resulting in extensive internalization and accumulation in tumour cells . DB06813 SUB is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies . Here , we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents . METHODS : Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay . Gene expression was evaluated using qRT-PCR . RESULTS : DB06813 SUB and methotrexate had a similar pattern of cytotoxicity , pralatrexate being more potent . DB06813 SUB potentiated the effects of platinum drugs , antimetabolites and P00533 REA inhibitors . Dose - and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of Q05932 REA . Acquired resistance to pralatrexate was associated with decreased P41440 REA - 1 expression , whereas methotrexate resistance correlated with increased P00374 REA expression , suggesting different mechanisms of acquired resistance . CONCLUSION : DB06813 SUB was more potent than methotrexate in a panel of solid tumour lines . Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies , and suggest that P41440 REA - 1 , Q05932 REA and P00374 REA may be potential biomarkers of outcome .