interacts_with DB00863?
Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in P13671 glioma cells : regulation by cyclic AMP . 1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived P13671 glioma cells have been investigated . 2 . P35367 -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 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 - P18146 - P05230 -GRL- P07550 -CS F1R- P09486 - P42261 - P29459 - P35916 -telomere ) was investigated in peripheral blood cell fractions . Gene dosage experiments demonstrated that P04141 , P18146 , P29459 , and P35916 were retained on the 5q- chromosome in both patients and that P05230 was retained in one patient , thus placing these genes outside the critical region . GRL , P07550 , P07333 , P09486 , and P42261 were shown to be deleted in both patients . The proximal breakpoint is localized between P18146 and P05230 in one patient and between P05230 and P07550 in the other , and the distal breakpoint is localized between P42261 and P29459 in both patients . Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints , and breakpoint-specific fragments were detected with P05230 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 and P29459 . O15379 represses the expression of P26718 ligands ULBPs in epithelial tumour cells : potential implications for the immunosurveillance of cancer . The expression of the P26718 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 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 ) , 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 . Furthermore , by small interfering RNA-mediated knockdown and overexpression of Q13547 -3 , we showed that O15379 is a repressor of ULBPs expression in epithelial cancer cells . Remarkably , P32119 treatment caused the complete release of O15379 from the Q9BZM6 -3 promoters . O15379 is recruited to the Q9BZM6 promoter through its interaction with Sp3 and P32119 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 ligands on human monocytic leukaemia THP-1 cells . Natural killer ( NK ) group 2D ( P26718 ) 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 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 ) 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 ligands , and this augmentation was significantly attenuated by histamine . The histamine H1 receptor ( P35367 ) agonist 2-pyridylethylamine and P25021 agonist dimaprit down-regulated the expression of P26718 ligands , and activation of P35367 and P25021 signalling by A23187 and forskolin , respectively , had the same effect , indicating that the histamine-induced down-regulation of P26718 ligands is mediated by P35367 and P25021 . Quantitative reverse transcription-PCR showed that mRNA levels of the P26718 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 . Furthermore , histamine treatment significantly reduced susceptibility to NK cell-mediated cytotoxicity . These results suggest that histamine down-regulates P26718 ligands through the activation of an P35367 - and P25021 -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 ) flavin domain , which has similar redox properties to those of NADPH-cytochrome P450 reductase ( P16435 ) , contains binding sites for calmodulin , DB03147 , 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 flavin domains , which include or delete the calmodulin ( P62158 ) -binding site . The air-stable semiquinone of the P29475 flavin domains showed similar redox properties to the corresponding DB03147 -FMNH ( & z.ccirf ; ) of P16435 . In the absence or presence of Ca(2+)/ P62158 , the rates of reduction of an DB03147 - 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 - 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 . [ Roles of P26718 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 receptors and corresponding ligands . The CIK cells was expanded from healthy individual with interferon (IFN)γ , CD3 monoclonal antibodies ( mAb ) and interleukin-2 ( P60568 ) . The subset of lymphocyte and the expression of NK cell receptors on CIK cells was detected by flow cytometry ; P26718 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 receptor but no other NK receptors such as CD158a , CD158b and NCR . Different levels of P26718 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 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;Daudi 68.67 ± 5.04 vs 52.67 ± 2.60 % , P = 0.024 ) . It is concluded that most of CIK cells express P26718 receptor , interaction of P26718 - P26718 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 ( 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 ) ; 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 cells with DB00544 , DB01629 , FUdR , DB00432 , 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 and Q9H3N8 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 agonists ( 4-methylhistamine , VUF8430 ) , P25021 agonist ( dimaprit ) and their combinations with Q9H3N8 antagonist ( JNJ10191584 ) and P25021 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-H2O2 based CL ) . KEY FINDINGS : DB11320 , 4-methylhistamine , VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner . On the other hand , only VUF8430 was able to inhibit PMA-activated whole blood CL . DB00863 , but not JNJ10191584 , 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 . Our results also suggest that Q9H3N8 agonists in concentrations higher than 10(-6)M may also influence ROS production via binding to P25021 . 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 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 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 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 , O95342 , P33527 , O15438 , O15440 , Q5T3U5 , and Q9UNQ0 in tumors may contribute to the generally poor treatment response of PDAC . The up-regulation of O95477 , Q8IZY2 , and P45844 implicates a serious impairment of cellular cholesterol homeostasis in PDAC . On the other hand , the observed down-regulation of Q99758 , O95255 , P13569 , and Q09428 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 ) , P25021 , Q9Y5N1 and Q9H3N8 . 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 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 ( P05112 ) , P05113 , P35225 , Interferon-gamma ( P01579 ) , tumor necrosis factor-alpha ( P01375 ) and P60568 in the BALF all increased significantly in both groups compared to those exposed to saline . In particular , the concentration of P01375 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 , 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 ) and histamine receptors . In contrast , the P35367 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 and P25021 . Therefore , P25103 and P25021 represent direct therapeutic targets for pain in IC/PBS and potentially other chronic pain conditions . Kir6.2-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 ( Kir6.2 ) which forms the channel pore , and the sulphonylurea receptor ( Q09428 ) , which serves as a regulatory subunit . The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure , and deletion of the N-terminus ( Kir6.2DeltaN14 ) results in functional uncoupling of the two subunits . In this study , we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with Q09428 and the effect of Kir6.2 on this interaction . We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of Q09428 and Kir6.2 in Kir6.2DeltaN14/ Q09428 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 alone , but with high affinity ( increased approximately 150-fold ) when Q09428 was co-expressed with Kir6.2 ( K(D)=0.42+/-0.03 nM ) . DB01016 , tolbutamide and nateglinide all bound with marginally lower affinity to Q09428 than to Kir6.2/ Q09428 . 3 . DB00912 bound with low affinity ( K(D)=51+/-23 nM ) to Q09428 co-expressed with Kir6.2DeltaN14 . 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 Kir6.2DeltaN14/ Q09428 currents by both repaglinide and nateglinde is abolished . 5 . The results suggest that Kir6.2 causes a conformational change in Q09428 required for high-affinity repaglinide binding , or that the high-affinity repaglinide-binding site includes contributions from both Q09428 and Kir6.2 . DB01016 , tolbutamide and nateglinide binding appear to involve only Q09428 . The design and development of pegfilgrastim ( PEG-rmetHuG- P04141 , 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 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 retains the same biological activity as filgrastim , and binds to the same Q99062 , 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 , 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 , which potently targets human Q13547 but also has inhibitory activity against Q92769 , O15379 , and Q96DB2 in vitro . In intact cells , DB05651 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal . DB05651 induced hyperacetylation of histones , selectively induced apoptosis , and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner . DB05651 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 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 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted . Mechanism of inhibition of the P42262 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 and its ( + ) counterpart about their mechanism of inhibition and selectivity toward four AMPA receptor subunits or P42261 -4 . We show that DB04982 is the eutomer with the endismic ratio being 14 for the closed-channel and 10 for the open-channel state of P42262 . Kinetic evidence supports that DB04982 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 . Given that DB04982 inhibits P42261 and P42262 , but is virtually ineffective on the P42263 and P48058 AMPA receptor subunits , we hypothesize that the " M " site(s) on P42261 and P42262 to which DB04982 binds is different from that on P42263 and P48058 . If the molecular properties of the AMPA receptors and DB04982 are used for selecting an inhibitor as a single drug candidate for controlling the activity of all AMPA receptors in vivo , DB04982 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 Dph2 , a [ 4Fe-4S ] enzyme required for diphthamide biosynthesis . DB03223 , the target of diphtheria toxin , is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 ( P13639 ) . 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 , PhDph2 , to catalyze the formation of a C-C bond . Crystal structure shows that PhDph2 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 PhDph2 . 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 PhDph2 dimer . DB00939 sodium is an inhibitor of both the P09917 and cyclooxygenase pathways of the arachidonic acid cascade in vitro . DB00939 sodium was compared to other nonsteroidal antiinflammatory drugs in terms of its potency to inhibit the formation of 5-HETE and LTB4 in human leukocytes and the formation of prostaglandin E2 in bovine seminal vesicles as measures of its ability to inhibit the P09917 and cyclooxygenase pathways of the arachidonic acid cascade . DB00939 sodium was about 2-4 times less potent than BW-755C in inhibiting P09917 enzyme activity and three times more potent than benoxaprofen , while naproxen , ibuprofen , and indomethacin showed IC50 greater than 100 microM . DB00939 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 sodium , like BW-755C , can be considered a dual inhibitor of P09917 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 ) on histamine metabolism in arteriosclerosis , the expression of histidine decarboxylase ( HDC ; histamine-producing enzyme ) , histamine receptors 1 and 2 ( P35367 and P25021 ) , and GM- P04141 was investigated in human and mouse arteriosclerotic carotid arteries . Furthermore , the molecular mechanisms of GM- P04141 -induced HDC and P35367 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 , P25021 , and GM- P04141 was also detected in the lesions . In U937 cells , GM- P04141 enhanced histamine secretion and gene expression of HDC and P35367 . A promoter assay showed that GM- P04141 enhanced gene transcription of HDC and P35367 but not P25021 . CONCLUSIONS : The present results indicate that HDC and HHR are expressed in arteriosclerotic lesion , and that GM- P04141 induces HDC and P35367 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 was demonstrated in arteriosclerotic lesions . In monocytic U937 cells , GM- P04141 upregulated the expression of histamine and P35367 . Coordinated expression of histamine and its receptors by GM- P04141 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 ) . 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 , 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 -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 -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 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 antagonist SR48968 and the selective P29371 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 to increase AngII-induced drinking , as well as water intake in sated or deprived rats suggests that brain P25103 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 DRB11302 and DQB10609 may be genetic markers for aspirin-induced urticaria/angioedema . A polymorphism study that examined nine single-nucleotide polymorphisms of five leukotriene-related genes [ P09917 ( encoding P09917 ) , P20292 ( P09917 -activating protein ) , P35354 ( cyclooxygenase 2 ) , Q16873 ( leukotriene C4 synthase ) , and Q9Y271 ( cysteinyl leukotriene receptor 1 ) ] found that promoter polymorphisms of P09917 ( -1708A > G ) and Q9Y271 ( -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 ( encoding histamine N-methyltransferase ) , P35367 or P25021 ( 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 DRB11302 and DQB10609 , and the P09917 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 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 , Q05925 , P09486 , P55290 and P25054 were analysed using melting curve analysis after DNA bisulfite treatment . P01116 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 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 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 .