MH_dev_181

Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in P13671 REA glioma cells : regulation by cyclic AMP . 1 . The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived P13671 REA glioma cells have been investigated . 2 . P35367 REA - stimulation caused a concentration-dependent increase in the accumulation of total [ 3H ] - inositol phosphates in cells prelabelled with [ 3H ] - myo-inositol . The rank order of agonist potencies was histamine ( EC50 = 24 microM ) > N alpha-methylhistamine ( EC50 = 31 microM ) > 2 - thiazolylethylamine ( EC50 = 91 microM ) . 3 . The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1 - antagonists , mepyramine ( apparent Kd = 1 nM ) and ( + ) - chlorpheniramine ( apparent Kd = 4 nM ) . In addition , ( - ) - chlorpheniramine was more than two orders of magnitude less potent than its ( + ) - stereoisomer . 4 . Elevation of intracellular cyclic AMP accumulation with forskolin ( 10 microM , EC50 = 0.3 microM ) , isoprenaline ( 1 microM , EC50 = 4 nM ) or rolipram ( 0.5 mM ) , significantly reduced the histamine-mediated ( 0.1 mM ) inositol phosphate response by 37 % , 43 % and 26 % respectively . In contrast , 1,9- dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine . 5 . These data indicate the presence of functionally coupled , endogenous histamine H1 receptors in P13671 REA glioma cells . Furthermore , the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells .

Molecular mapping of uncharacteristically small 5q deletions in two patients with the 5q - syndrome : delineation of the critical region on 5q and identification of a 5q - breakpoint . Molecular mapping techniques have defined the region of gene loss in two patients with the 5q - syndrome and uncharacteristically small 5q deletions ( 5q31 - q33 ) . The allelic loss of 10 genes localized to 5q23 - qter ( centromere - P04141 REA - P18146 REA - P05230 REA - GRL - P07550 REA - CS F1R - P09486 REA - P42261 REA - P29459 REA - P35916 REA - telomere ) was investigated in peripheral blood cell fractions . Gene dosage experiments demonstrated that P04141 REA , P18146 REA , P29459 REA , and P35916 REA were retained on the 5q - chromosome in both patients and that P05230 REA was retained in one patient , thus placing these genes outside the critical region . GRL , P07550 REA , P07333 REA , P09486 REA , and P42261 REA were shown to be deleted in both patients . The proximal breakpoint is localized between P18146 REA and P05230 REA in one patient and between P05230 REA and P07550 REA in the other , and the distal breakpoint is localized between P42261 REA and P29459 REA in both patients . Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints , and breakpoint-specific fragments were detected with P05230 REA in the granulocyte but not the lymphocyte fraction of one patient . This study has established the critical region of gene loss of the 5q - chromosome in the 5q - syndrome , giving the location for a putative tumor-suppressor gene in the 5.6- Mb region between P05230 REA and P29459 REA .

O15379 REA represses the expression of P26718 REA ligands ULBPs in epithelial tumour cells : potential implications for the immunosurveillance of cancer . The expression of the P26718 REA ligands on cancer cells leads to their recognition and elimination by host immune responses mediated by natural killer and T cells . UL16 - binding proteins ( ULBPs ) are P26718 REA ligands , which are scarcely expressed in epithelial tumours , favouring their evasion from the immune system . Herein , we investigated the epigenetic mechanisms underlying the repression of ULBPs in epithelial cancer cells . We show that Q9BZM6 - 3 expression is increased in tumour cells after exposure to the inhibitor of histone deacetylases ( HDACs ) trichostatin A ( P32119 REA ) , which enhances the natural killer cell-mediated cytotoxicity of HeLa cells . Our experiments showed that the transcription factor Sp3 is crucial in the activation of the Q9BZM6 promoter by P32119 REA . Furthermore , by small interfering RNA-mediated knockdown and overexpression of Q13547 REA - 3 , we showed that O15379 REA is a repressor of ULBPs expression in epithelial cancer cells . Remarkably , P32119 REA treatment caused the complete release of O15379 REA from the Q9BZM6 - 3 promoters . O15379 REA is recruited to the Q9BZM6 promoter through its interaction with Sp3 and P32119 REA treatment interfered with this association . Together , we describe a new mechanism by which cancer cells may evade the immune response through the epigenetic modulation of the ULBPs expression and provide a model in which HDAC inhibitors may favour the elimination of transformed cells by increasing the immunogenicity of epithelial tumours .

DB11320 reduces susceptibility to natural killer cells via down-regulation of P26718 REA ligands on human monocytic leukaemia THP - 1 cells . Natural killer ( NK ) group 2D ( P26718 REA ) is a key activating receptor expressed on NK cells , whose interaction with ligands on target cells plays an important role in tumorigenesis . However , the effect of histamine on P26718 REA ligands on tumour cells is unclear . Here we showed that human monocytic leukaemia THP - 1 cells constitutively express MHC class I-related chain A ( Q29983 REA ) and Q9BZM6 on their surface , and incubation with histamine reduced the expression in a dose-dependent and time-dependent manner as assessed by flow cytometry . Interferon-γ augmented the surface expression of the P26718 REA ligands , and this augmentation was significantly attenuated by histamine . The histamine H1 receptor ( P35367 REA ) agonist 2 - pyridylethylamine and P25021 REA agonist dimaprit down-regulated the expression of P26718 REA ligands , and activation of P35367 REA and P25021 REA signalling by A23187 and forskolin , respectively , had the same effect , indicating that the histamine-induced down-regulation of P26718 REA ligands is mediated by P35367 REA and P25021 REA . Quantitative reverse transcription-PCR showed that mRNA levels of the P26718 REA ligands and relevant microRNAs were not significantly changed by histamine . DB11320 down-regulated the surface expression of endoplasmic reticulum protein 5 , and inhibition of matrix metalloproteinases did not impair this down-regulation , indicating that proteolytic shedding was not involved . Instead , pharmacological inhibition of protein transport and proteasome abrogated it , and histamine enhanced ubiquitination of Q29983 REA . Furthermore , histamine treatment significantly reduced susceptibility to NK cell-mediated cytotoxicity . These results suggest that histamine down-regulates P26718 REA ligands through the activation of an P35367 REA - and P25021 REA - mediated ubiquitin-proteasome pathway and consequently reduces susceptibility to NK cells .

P62158 activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain . The neuronal NO synthase ( P29475 REA ) flavin domain , which has similar redox properties to those of NADPH-cytochrome P450 reductase ( P16435 REA ) , contains binding sites for calmodulin , DB03147 MEN , Q68DA7 , and NADPH . The aim of this study is to elucidate the mechanism of activation of the flavin domain by calcium / calmodulin ( Ca ( 2 + ) / P62158 ) . In this study , we used the recombinant P29475 REA flavin domains , which include or delete the calmodulin ( P62158 ) - binding site . The air-stable semiquinone of the P29475 REA flavin domains showed similar redox properties to the corresponding DB03147 MEN - FMNH ( & z.ccirf ; ) of P16435 REA . In the absence or presence of Ca ( 2 + ) / P62158 , the rates of reduction of an DB03147 MEN - Q68DA7 pair by NADPH have been investigated at different wavelengths , 457 , 504 and 590 nm by using a stopped-flow technique and a rapid scan spectrophotometry . The reduction of the oxidized enzyme ( DB03147 MEN - Q68DA7 ) by NADPH proceeds by both one-electron equivalent and two-electron equivalent mechanisms , and the formation of semiquinone ( increase of absorbance at 590 nm ) was significantly increased in the presence of Ca ( 2 + ) / P62158 . The air-stable semiquinone form of the enzyme was also rapidly reduced by NADPH . The results suggest that an intramolecular one-electron transfer between the two flavins is activated by the binding of Ca ( 2 + ) / P62158 . The F ( 1 ) H ( 2 ) , which is the fully reduced form of the air-stable semiquinone , can donate one electron to the electron acceptor , cytochrome c . The proposed mechanism of activation by Ca ( 2 + ) / P62158 complex is discussed on the basis of that provided by P16435 REA .

[ Roles of P26718 REA in cytokine-induced killer ( CIK ) against hematological malignant cells lines ] . This study was purposed to investigate the CIK cell cytotoxicity to hematological malignant cell lines by interaction P26718 REA receptors and corresponding ligands . The CIK cells was expanded from healthy individual with interferon ( IFN ) γ , CD3 monoclonal antibodies ( mAb ) and interleukin - 2 ( P60568 REA ) . The subset of lymphocyte and the expression of NK cell receptors on CIK cells was detected by flow cytometry ; P26718 REA ligand expression on hematological malignant cell lines was also analyzed by flow cytometry , the calcein acetoxymethyl ester ( P62158 ) was used for labeling target cells , then the cytotoxicity of CIK cells to hematological malignant cell lines was detected by flow cytometry . The results showed that most of CIK cells expressed CD3 ( 97.85 ± 1.95 % ) , CD3 ( + ) CD8 ( + ) cells and CD3 ( + ) CD56 ( + ) cells increased significantly as compared with un-cultured cells ( P < 0.001 ;P = 0.033 ) . About 86 % CIK cells expressed P26718 REA receptor but no other NK receptors such as CD158a , CD158b and NCR . Different levels of P26718 REA ligands were detected in hematological malignant cell lines U266 , K562 and Daudi . CIK cells showed high cytotoxicity to these three different cell lines , and this cytotoxicity was partially blocked by treating CIK cells with anti - P26718 REA antibody ( U266 52.67 ± 4.63 % vs 32.67 ± 4.81 % , P = 0.008 ; K562 71.67 ± 4.91 % vs 50.33 ± 4.91 % , P = 0.007 ;D audi 68.67 ± 5.04 vs 52.67 ± 2.60 % , P = 0.024 ) . It is concluded that most of CIK cells express P26718 REA receptor , interaction of P26718 REA - P26718 REA ligands may be one of the mechanisms , by which CIK cells kill hematological malignant cells .

Detection of thymidylate synthase modulators by a novel screening assay . P04818 REA ( TS ) , a key cancer chemotherapeutic target , catalyzes the conversion of deoxyuridylate to thymidylate . TS can serve as a repressor of its own synthesis by binding to its own mRNA through TS-specific binding elements ( TBEs ) . In this report , we describe the use of a luciferase reporter plasmid containing two TBEs that can be used as a tool for the identification and initial profiling of compounds that modulate TS activity , levels , or ability to bind mRNA . To validate this model , we evaluated several groups of drugs . Thus , cells were exposed to the pyrimidine analogs 5 - fluorouracil ( DB00544 ) , 5 - fluorouridine ( DB01629 ) , 5 - fluoro - 2 ' - deoxyuridine ( FUdR ) , trifluorothymidine ( DB00432 MEN ) ; to the nonpyrimidine TS-inhibitors AG - 331 , nolatrexed ( AG337 ) , and raltitrexed ( DB00293 ) ; or to drugs with other primary sites of action ( methotrexate , actinomycin D , 5 - azacytidine , 8 - thioguanosine ) . Except for 5 - azacytidine and 8 - thioguanosine , all compounds examined induced luciferase activity compared with untreated cells . Effects of luciferase activity inducing drugs through TS-affected translation were confirmed by examinations of TS protein and mRNA levels . Treatment of H630 - P13671 REA cells with DB00544 , DB01629 , FUdR , DB00432 MEN , AG331 , AG337 , DB00293 , and methotrexate up-regulated TS levels as determined by Western blot analysis , although TS mRNA levels remained unchanged as determined by reverse transcription-polymerase chain reaction . Our studies demonstrate a novel application of a TBE-dependent reporter plasmid that could be used for the high-throughput identification of potential chemotherapeutic agents that modulate TS RNA-binding activity , either directly or indirectly .

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes . AIMS : The diverse physiological functions of histamine are mediated through distinct histamine receptors . In this study we investigated the role of P25021 REA and Q9H3N8 REA in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood . MAIN METHODS : Changes in reactive oxygen species ( ROS ) production by whole blood phagocytes after treatment with histamine , Q9H3N8 REA agonists ( 4 - methylhistamine , VUF 8430 ) , P25021 REA agonist ( dimaprit ) and their combinations with Q9H3N8 REA antagonist ( JNJ 10191584 ) and P25021 REA antagonist ( ranitidine ) were determined using the chemiluminescence ( CL ) assay . To exclude the direct scavenging effects of the studied compounds on the CL response , the antioxidant properties of all compounds were measured using several methods ( TRAP , ORAC , and luminol-HRP-H 2O2 based CL ) . KEY FINDINGS : DB11320 , 4 - methylhistamine , VUF 8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner . On the other hand , only VUF 8430 was able to inhibit PMA-activated whole blood CL . DB00863 SUB , but not JNJ 10191584 , completely reduced the effects of histamine , 4 - methylhistamine and dimaprit . The direct scavenging ability of tested compounds was negligible . SIGNIFICANCE : Our results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by P25021 REA . Our results also suggest that Q9H3N8 REA agonists in concentrations higher than 10 ( - 6 ) M may also influence ROS production via binding to P25021 REA .

Differences in transcript levels of ABC transporters between pancreatic adenocarcinoma and nonneoplastic tissues . OBJECTIVES : The aim of this study was to evaluate transcript levels of all 49 human DB00171 - binding cassette transporters ( ABCs ) in one of the most drug-resistant cancers , namely , the pancreatic ductal adenocarcinoma ( PDAC ) . Association of ABCs levels with clinical-pathologic characteristics and P01116 REA mutation status was followed as well . METHODS : Tumors and adjacent nonneoplastic tissues were obtained from 32 histologically verified PDAC patients . The transcript profile of ABCs was assessed using quantitative real-time polymerase chain reaction with a relative standard curve . P01116 REA mutations in exon 2 were assessed by high-resolution melting analysis and sequencing . RESULTS : Most ABCs were deregulated in PDAC and 10 ABCs were associated with clinical-pathologic characteristics . P01116 REA mutations did not change the global expression profile of ABCs . CONCLUSIONS : The expression of ABC transporters was significantly deregulated in PDAC tumors when compared to nonmalignant tissues . The observed up-regulation of P21439 REA , O95342 REA , P33527 REA , O15438 REA , O15440 REA , Q5T3U5 REA , and Q9UNQ0 in tumors may contribute to the generally poor treatment response of PDAC . The up-regulation of O95477 REA , Q8IZY2 , and P45844 REA implicates a serious impairment of cellular cholesterol homeostasis in PDAC . On the other hand , the observed down-regulation of Q99758 REA , O95255 REA , P13569 REA , and Q09428 REA suggests a possible role of stem cells in the development and progression of PDAC .

Enhanced goblet cell hyperplasia in HDC knockout mice with allergic airway inflammation . BACKGROUND : DB11320 is known to have immunoregulatory roles in allergic reactions through histamine receptor 1 ( P35367 REA ) , P25021 REA , Q9Y5N1 REA and Q9H3N8 REA . However , its role in goblet cell hyperplasia in the airways of asthma patients is yet to be clarified . OBJECTIVE : This study was designed to examine the role of histamine in goblet cell hyperplasia using histamine-deficient mice ( Hdc - / - mice ) with allergic airway inflammation . METHODS : Wild-type and Hdc - / - C57BL / 6 mice were sensitized with ovalbumin ( OVA ) . After a 2 - week exposure to OVA , goblet cell hyperplasia was evaluated . Cell differentials and cytokines in BALF were analyzed . The mRNA levels of P98088 REA and Gob - 5 gene were determined quantitatively . RESULTS : The number of eosinophils in BALF increased in both the sensitized wild-type mice and Hdc - / - mice with OVA inhalation . In addition , the numbers of alveolar macrophages and lymphocytes in BALF increased significantly in the sensitized Hdc - / - mice with OVA inhalation compared to the wild-type mice under the same conditions . The concentrations of P05112 REA ( P05112 REA ) , P05113 REA , P35225 REA , Interferon-gamma ( P01579 REA ) , tumor necrosis factor-alpha ( P01375 REA ) and P60568 REA in the BALF all increased significantly in both groups compared to those exposed to saline . In particular , the concentration of P01375 REA in the Hdc - / - mice exposed to OVA was significantly higher than that in the wild-type mice under the same conditions . The mRNA levels of Gob - 5 and P98088 REA , and the ratio of the goblet cells in the airway epithelium significantly increased in Hdc - / - mice exposed to OVA compared to wild-type mice . CONCLUSIONS : These results suggested that histamine may play a regulatory role in goblet cell hyperplasia in allergic airway inflammation .

Pharmacologic attenuation of pelvic pain in a murine model of interstitial cystitis . BACKGROUND : Interstitial cystitis / painful bladder syndrome ( IC / PBS ) is a bladder disease that causes debilitating pelvic pain of unknown origin , and IC / PBS symptoms correlate with elevated bladder lamina propria mast cell counts . Similar to IC / PBS patients , pseudorabies virus ( PRV ) infection in mice induces a neurogenic cystitis associated with bladder lamina propria mast cell accumulation and pelvic pain . We evaluated several drugs to determine the effectiveness of reducing PRV-induced pelvic pain . METHODS : Neurogenic cystitis was induced by the injection of Bartha ' s strain of PRV into the abductor caudalis dorsalis tail base muscle of female C57BL / 6 mice . Therapeutic modulation of pelvic pain was assessed daily for five days using von Frey filament stimulation to the pelvic region to quantify tactile allodynia . RESULTS : Significant reduction of PRV-induced pelvic pain was observed for animals treated with antagonists of neurokinin receptor 1 ( P25103 REA ) and histamine receptors . In contrast , the P35367 REA antagonist hydroxyzine , proton pump inhibitors , a histamine receptor 3 agonist , and gabapentin had little or no effect on PRV-induced pelvic pain . CONCLUSION : These data demonstrate that bladder-associated pelvic pain is attenuated by antagonists of P25103 REA and P25021 REA . Therefore , P25103 REA and P25021 REA represent direct therapeutic targets for pain in IC / PBS and potentially other chronic pain conditions .

Kir 6.2- dependent high-affinity repaglinide binding to beta-cell K ( DB00171 ) channels . 1 . The beta-cell K ( DB00171 ) channel is composed of two types of subunit - the inward rectifier K ( + ) channel ( Kir 6.2 ) which forms the channel pore , and the sulphonylurea receptor ( Q09428 REA ) , which serves as a regulatory subunit . The N-terminus of Kir 6.2 is involved in transduction of sulphonylurea binding into channel closure , and deletion of the N-terminus ( Kir 6.2 DeltaN 14 ) results in functional uncoupling of the two subunits . In this study , we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with Q09428 REA and the effect of Kir 6.2 on this interaction . We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of Q09428 REA and Kir 6.2 in Kir 6.2 DeltaN 14 / Q09428 REA channels . All binding experiments are performed on membranes in DB00171 - free buffer at 37 degrees C . 2 . DB00912 was found to bind with low affinity ( K ( D )= 59 + / - 16 nM ) to Q09428 REA alone , but with high affinity ( increased approximately 150 - fold ) when Q09428 REA was co-expressed with Kir 6.2 ( K ( D )= 0.42+ / -0.03 nM ) . DB01016 MENMAX DB01016 MEN , tolbutamide and nateglinide all bound with marginally lower affinity to Q09428 REA than to Kir 6.2 / Q09428 REA . 3 . DB00912 bound with low affinity ( K ( D )= 51 + / - 23 nM ) to Q09428 REA co-expressed with Kir 6.2 DeltaN 14 . In contrast , the affinity for glibenclamide , tolbutamide and nateglinide was only mildly changed as compared to wild-type channels . 4 . In whole-cell patch-clamp experiments inhibition of Kir 6.2 DeltaN 14 / Q09428 REA currents by both repaglinide and nateglinde is abolished . 5 . The results suggest that Kir 6.2 causes a conformational change in Q09428 REA required for high-affinity repaglinide binding , or that the high-affinity repaglinide-binding site includes contributions from both Q09428 REA and Kir 6.2 . DB01016 MEN , tolbutamide and nateglinide binding appear to involve only Q09428 REA .

The design and development of pegfilgrastim ( PEG-rmetHuG - P04141 REA , Neulasta ) . Recombinant protein technology produces drugs for human therapy in unprecedented quantity and quality . Research is now focusing on the relationship between pharmacokinetic and pharmacodynamic properties of molecules , with the aim of engineering proteins that possess enhanced therapeutic characteristics in contrast to being used as simple replacements for the natural equivalent . The addition of a polyethylene glycol ( PEG ) moiety to filgrastim ( rmetHu - DB00099 , Neupogen ) resulted in the development of pegfilgrastim . DB00019 MEN is a long-acting form of filgrastim that requires only once-per-cycle administration for the management of chemotherapy-induced neutropenia . The covalent attachment of PEG to the N-terminal amine group of the parent molecule was attained using site-directed reductive alkylation . Pegylation increases the size of filgrastim so that it becomes too large for renal clearance . Consequently , neutrophil-mediated clearance predominates in elimination of the drug . This extends the median serum half-life of pegfilgrastim to 42 hours , compared with between 3.5 and 3.8 hours for DB00099 , though in fact the half-life is variable , depending on the absolute neutrophil count , which in turn reflects of the ability of pegfilgrastim to sustain production of those same cells . The clearance of the molecule is thus dominated by a self-regulating mechanism . DB00019 MEN retains the same biological activity as filgrastim , and binds to the same Q99062 REA , stimulating the proliferation , differentiation and activation of neutrophils . Once-per-chemotherapy cycle administration of pegfilgrastim reduces the duration of severe neutropenia as effectively as daily treatment with filgrastim . In clinical trials , patients receiving pegfilgrastim also had a lower observed incidence of febrile neutropenia than patients receiving filgrastim .

DB05651 MEN , a novel isotype-selective histone deacetylase inhibitor , has broad spectrum antitumor activity in vitro and in vivo . Nonselective inhibitors of human histone deacetylases ( HDAC ) are known to have antitumor activity in mice in vivo , and several of them are under clinical investigation . The first of these , DB02546 ( DB02546 ) , has been approved for treatment of cutaneous T-cell lymphoma . Questions remain concerning which HDAC isotype ( s ) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotype-selective HDAC inhibitor . We have developed an isotype-selective HDAC inhibitor , DB05651 MEN , which potently targets human Q13547 REA but also has inhibitory activity against Q92769 REA , O15379 REA , and Q96DB2 in vitro . In intact cells , DB05651 MEN inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal . DB05651 MEN induced hyperacetylation of histones , selectively induced apoptosis , and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner . DB05651 MEN exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro , and HDAC inhibitory activity was required for these effects . In vivo , DB05651 MEN significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors . Our findings suggest that the isotype-selective HDAC inhibition by DB05651 MEN is sufficient for antitumor activity in vivo and that further clinical investigation is warranted .

Mechanism of inhibition of the P42262 REA AMPA receptor channel opening by talampanel and its enantiomer : the stereochemistry of the 4 - methyl group on the diazepine ring of 2,3- benzodiazepine derivatives . Stereoselectivity of 2,3- benzodiazepine compounds provides a unique way for the design of stereoisomers as more selective and more potent inhibitors as drug candidates for treatment of the neurological diseases involving excessive activity of AMPA receptors . Here we investigate a pair of enantiomers known as DB04982 MEN and its ( + ) counterpart about their mechanism of inhibition and selectivity toward four AMPA receptor subunits or P42261 REA - 4 . We show that DB04982 MEN is the eutomer with the endismic ratio being 14 for the closed-channel and 10 for the open-channel state of P42262 REA . Kinetic evidence supports that DB04982 MEN is a noncompetitive inhibitor and it binds to the same site for those 2,3- benzodiazepine compounds with the C - 4 methyl group on the diazepine ring . This site , which we term as the " M " site , recognizes preferentially those 2,3- benzodiazepine compounds with the C - 4 methyl group being in the R configuration , as in the chemical structure of DB04982 MEN . Given that DB04982 MEN inhibits P42261 REA and P42262 REA , but is virtually ineffective on the P42263 REA and P48058 REA AMPA receptor subunits , we hypothesize that the " M " site ( s ) on P42261 REA and P42262 REA to which DB04982 MEN binds is different from that on P42263 REA and P48058 REA . If the molecular properties of the AMPA receptors and DB04982 MEN are used for selecting an inhibitor as a single drug candidate for controlling the activity of all AMPA receptors in vivo , DB04982 MEN is not ideal . Our results further suggest that addition of longer acyl groups to the N - 3 position should produce more potent 2,3- benzodiazepine inhibitors for the " M " site .

Mechanistic understanding of Pyrococcus horikoshii Dph 2 , a [ 4Fe - 4S ] enzyme required for diphthamide biosynthesis . DB03223 MEN , the target of diphtheria toxin , is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 ( P13639 REA ) . The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii ( P . horikoshii ) , the first step uses an DB00118 ( DB00118 ) - dependent [ 4Fe - 4S ] enzyme , PhDph 2 , to catalyze the formation of a C-C bond . Crystal structure shows that PhDph 2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [ 4Fe - 4S ] cluster . In the reduced state , the [ 4Fe - 4S ] cluster can provide one electron to reductively cleave the bound DB00118 molecule . However , different from classical radical DB00118 family of enzymes , biochemical evidence suggest that a 3 - amino - 3 - carboxypropyl radical is generated in PhDph 2 . Here we present evidence supporting that the 3 - amino - 3 - carboxypropyl radical does not undergo hydrogen abstraction reaction , which is observed for the deoxyadenosyl radical in classical radical DB00118 enzymes . Instead , the 3 - amino - 3 - carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product . Furthermore , our data suggest that the chemistry requires only one [ 4Fe - 4S ] cluster to be present in the PhDph 2 dimer .

DB00939 MEN sodium is an inhibitor of both the P09917 REA and cyclooxygenase pathways of the arachidonic acid cascade in vitro . DB00939 MEN sodium was compared to other nonsteroidal antiinflammatory drugs in terms of its potency to inhibit the formation of 5 - HETE and LTB 4 in human leukocytes and the formation of prostaglandin E2 in bovine seminal vesicles as measures of its ability to inhibit the P09917 REA and cyclooxygenase pathways of the arachidonic acid cascade . DB00939 MEN sodium was about 2-4 times less potent than BW - 755C in inhibiting P09917 REA enzyme activity and three times more potent than benoxaprofen , while naproxen , ibuprofen , and indomethacin showed IC50 greater than 100 microM . DB00939 MEN sodium and indomethacin were the most potent inhibitors of the formation of DB00917 in bovine seminal vesicles followed by ibuprofen , naproxen , and benoxaprofen in this order . DB00939 MEN sodium , like BW - 755C , can be considered a dual inhibitor of P09917 REA and cyclooxygenase pathways of arachidonic acid cascade . This finding may explain in part the antiinflammatory activity of meclofenamate sodium .

Granulocyte macrophage-colony stimulating factor increases the expression of histamine and histamine receptors in monocytes / macrophages in relation to arteriosclerosis . OBJECTIVE : To study the effect of granulocyte macrophage-colony-stimulating factor ( GM - P04141 REA ) on histamine metabolism in arteriosclerosis , the expression of histidine decarboxylase ( HDC ; histamine-producing enzyme ) , histamine receptors 1 and 2 ( P35367 REA and P25021 REA ) , and GM - P04141 REA was investigated in human and mouse arteriosclerotic carotid arteries . Furthermore , the molecular mechanisms of GM - P04141 REA - induced HDC and P35367 REA expression in monocytic U937 cells were investigated . METHODS AND RESULTS : Immunohistochemistry showed that atherosclerotic human coronary and mouse ligated carotid arteries contained HDC-expressing macrophages . Gene expression of HDC , P35367 REA , P25021 REA , and GM - P04141 REA was also detected in the lesions . In U937 cells , GM - P04141 REA enhanced histamine secretion and gene expression of HDC and P35367 REA . A promoter assay showed that GM - P04141 REA enhanced gene transcription of HDC and P35367 REA but not P25021 REA . CONCLUSIONS : The present results indicate that HDC and HHR are expressed in arteriosclerotic lesion , and that GM - P04141 REA induces HDC and P35367 REA expression in monocytes . Locally produced histamine might participate in atherogenesis by affecting the expression of atherosclerosis-related genes in monocytes and smooth muscle cells . The presence of histamine-producing macrophages and gene expression of histamine receptors and GM - P04141 REA was demonstrated in arteriosclerotic lesions . In monocytic U937 cells , GM - P04141 REA upregulated the expression of histamine and P35367 REA . Coordinated expression of histamine and its receptors by GM - P04141 REA would participate in atherogenesis by affecting monocytic and SMC gene expression .

Engineering toxin-resistant therapeutic stem cells to treat brain tumors . Pseudomonas exotoxin ( PE ) potently blocks protein synthesis by catalyzing the inactivation of elongation factor - 2 ( P13639 REA ) . Targeted PE-cytotoxins have been used as antitumor agents , although their effective clinical translation in solid tumors has been confounded by off-target delivery , systemic toxicity , and short chemotherapeutic half-life . To overcome these limitations , we have created toxin-resistant stem cells by modifying endogenous P13639 REA , and engineered them to secrete PE-cytotoxins that target specifically expressed ( interleukin - 13 receptor subunit alpha - 2 ) or overexpressed ( epidermal growth factor receptor ) in glioblastomas ( GBM ) . Molecular analysis correlated efficacy of PE-targeted cytotoxins with levels of cognate receptor expression , and optical imaging was applied to simultaneously track the kinetics of protein synthesis inhibition and GBM cell viability in vivo . The release of P35225 REA - PE from biodegradable synthetic extracellular matrix ( sECM ) encapsulated stem cells in a clinically relevant GBM resection model led to increased long-term survival of mice compared to P35225 REA - PE protein infusion . Moreover , multiple patient-derived GBM lines responded to treatment , underscoring its clinical relevance . In sum , integrating stem cell-based engineering , multimodal imaging , and delivery of PE-cytotoxins in a clinically relevant GBM model represents a novel strategy and a potential advancement in GBM therapy .

Further evidence that central tachykinin NK - 1 receptors mediate the inhibitory effect of tachykinins on angiotensin-induced drinking in rats . The order of potency of tachykinin ( TK ) receptor agonists suggests that TK NK - 1 receptors mediate their inhibitory effect on water intake induced by intracerebroventricular ( i . c . v . ) injection of angiotensin II ( AngII ) in rats . The present study was aimed at further evaluating which TK receptor subtype mediates the effect , using selective antagonists for the TK receptor subtypes . Pulse i . c . v . injection of the TK agonist neuropeptide gamma ( NP gamma ) , 31-250 ng / rat , markedly inhibited AngII-induced water intake . The i . c . v . injection of the P25103 REA antagonist SR14033 , 0.5 microgram / rat , significantly reduced , while 1 microgram / rat completely abolished the inhibitory effect of NP gamma , 125 ng / rat . The selective P21452 REA antagonist SR48968 and the selective P29371 REA antagonist R820 were devoid of any effect up to the i . c . v . dose of 2 micrograms / rat . On the other hand , i . c . v . injection of SR140333 , 1 microgram / rat , did not increase drinking induced by i . c . v . injection of AngII , 0.1- 10 ng / rat , and did not increase drinking in water sated or water deprived rats . The results of the present study confirm that central TKergic mechanisms inhibit AngII-induced drinking in rats , and provide further evidence that TK NK - 1 receptors mediate the effect . Failure of i . c . v . injected DB05790 MEN to increase AngII-induced drinking , as well as water intake in sated or deprived rats suggests that brain P25103 REA mechanisms apparently do not exert a tonic control on AngII-induced drinking and , in general , on water intake in rats . From a pharmacological point of view , the inhibitory effect of TKs on the dipsogenic action of AngII can represent a functional test for activity at central NK - 1 receptors in rats .

Genetic mechanism of aspirin-induced urticaria / angioedema . PURPOSE OF REVIEW : DB00945 - induced urticaria / angioedema is a major aspirin-related hypersensitivity often associated with aspirin-intolerant asthma . Genetic studies on aspirin-intolerant asthma have shown chronic overproduction of cysteinyl leukotrienes . The genetic analysis of aspirin-induced urticaria / angioedema is limited , however . RECENT FINDINGS : A recent study on HLA genotypes has suggested that the HLA alleles DRB 11302 and DQB 10609 may be genetic markers for aspirin-induced urticaria / angioedema . A polymorphism study that examined nine single-nucleotide polymorphisms of five leukotriene-related genes [ P09917 REA ( encoding P09917 REA ) , P20292 REA ( P09917 REA - activating protein ) , P35354 REA ( cyclooxygenase 2 ) , Q16873 REA ( leukotriene C4 synthase ) , and Q9Y271 REA ( cysteinyl leukotriene receptor 1 ) ] found that promoter polymorphisms of P09917 REA ( - 1708A > G ) and Q9Y271 REA ( - 634C > T ) were significantly different between aspirin-intolerant asthma and aspirin-induced urticaria / angioedema , suggesting different contributions to the lipoxygenase pathway . A second polymorphism study , conducted on histamine-related genes , did not find any significant associations with aspirin-induced urticaria / angioedema for the genes P50135 REA ( encoding histamine N-methyltransferase ) , P35367 REA or P25021 REA ( encoding histamine receptor types 1 and 2 respectively ) , or the gene encoding high-affinity IgE receptor Ibeta ( FcepsilonRIbeta ) ; however , the FcepsilonRIalpha gene promoter polymorphism was significantly associated with aspirin-induced urticaria / angioedema . This finding has been supported by in vitro functional studies . SUMMARY : The HLA alleles DRB 11302 and DQB 10609 , and the P09917 REA and FcepsilonRIalpha promoter polymorphisms , may contribute to the pathogenesis of aspirin-induced urticaria / angioedema . Further investigation to identify candidate genetic markers would help to elucidate the pathogenic mechanism of this condition .

Genetic and epigenetic markers in the evaluation of pancreatic masses . BACKGROUND : Methylation markers have shown promise in the early diagnosis of pancreatic carcinoma . The aim of this study was to assess the diagnostic utility of hypermethylation status of candidate genes in combination with P01116 REA mutation detection in the evaluation of pancreatic masses . EXPERIMENTAL DESIGN : Sixty-one fine needle aspirates of pancreatic masses ( 43 pancreatic adenocarcinomas and 18 chronic pancreatitis ) were studied . Methylation status of P25021 REA , Q05925 , P09486 REA , P55290 and P25054 REA were analysed using melting curve analysis after DNA bisulfite treatment . P01116 REA mutations were also analysed . RESULTS : The methylation panel had a sensitivity of 73 % ( 27 of 37 , CI 95 % 56 to 86 % ) and a specificity of 100 % whenever two or more promoters were found hypermethylated . P01116 REA mutations showed a sensitivity of 77 % ( 33 of 43 , CI 95 % 62 to 88 % ) and a specificity of 100 % . Both molecular analyses added useful information to cytology by increasing the number of informative cases . When genetic and epigenetic analyses were combined sensitivity was 84 % ( 36 of 43 CI 95 % 69 to 93 % ) maintaining a 100 % specificity . CONCLUSIONS : Analysis of hypermethylation status of a panel of genes and P01116 REA mutation detection offer a similar diagnostic yield in the evaluation of pancreatic masses . The combined molecular analysis increases the number of informative cases without diminishing specificity .