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[ "DB00072", "DB00294", "DB00338", "DB00341", "DB00588", "DB00820", "DB02546", "DB02901", "DB04844" ]
Induction of apoptosis of Beta cells of the pancreas by advanced glycation end-products , important mediators of chronic complications of diabetes mellitus . We herein report cytotoxicity of advanced glycation end-products ( AGEs ) on pancreatic beta cells . AGEs stimulated reactive oxygen species ( ROS ) generation but did not arrest proliferation of the P01308 -1 cell line . Pancreatic beta cell lines or primary cultured islets possess a receptor for P51606 ( RAGE ) , and its expression increased after P51606 treatment . TUNEL staining and FACS analysis using annexin V/PI antibodies showed that apoptosis increased in P01308 -1 cells or primary cultured islets when incubated with BSA conjugated with glyceraldehyde ( AGE2 ) or glucoaldehyde ( AGE3 ) , compared with those conjugated with glucose ( AGE1 ) . Reaction of P01308 -1 cells to Ki67 , which is a cellular marker for proliferation , was also increased after P51606 treatment . The ability of primary cultured islets to secrete insulin was retained even after P51606 treatment under either low or high glucose conditions . The antiserum against RAGE partially prevented P51606 -induced cellular events . Treatment of beta cells with the antioxidant metallothionein results in a significant reduction in pathologic changes . AGEs might be able to induce apoptosis as well as proliferation of pancreatic beta cell lines or primary cultured islets . Moreover , antibody array showed that Q06609 and P43351 were significantly decreased in AGE2-treated P01308 -1 cells . AGEs might inhibit homologous DNA recombination for repairing DNA of P01308 -1 cells damaged by ROS generation . It might be suggested that treatment of AGEs resulted in ROS production and apoptosis through their receptor on pancreatic beta cells . AGEs might deteriorate function of pancreatic beta cells in patients with long-term hyperglycemia . Chronic daily tadalafil prevents the corporal fibrosis and veno-occlusive dysfunction that occurs after cavernosal nerve resection . OBJECTIVES : To determine whether a long-term single daily oral dose of a longer half-life phosphodiesterase-5 ( O76074 ) inhibitor , tadalafil , has a similar effect to that of the shorter half-life O76074 inhibitors sildenafil and vardenafil , and can prevent the fibrosis and resultant corporal veno-occlusive dysfunction ( CVOD ) occurring after cavernosal nerve ( CN ) injury . MATERIALS AND METHODS : Male rats ( 10 per group ) had either a sham operation , unilateral CN resection ( P21554 ) or bilateral P21554 , and were left untreated or given retrolingually 5 mg/kg per day of tadalafil . After 45 days , CVOD was assessed via cavernosometry , and the underlying corporal tissue changes were examined by immunohistochemistry and histochemistry ( followed by quantitative image analysis ) , Western blots , and ad hoc methods . RESULTS : DB00820 treatment normalized the low response to papaverine and high drop rate in the intracavernosal pressure measured by cavernosometry after P21554 compared with sham-operated rats . DB00820 also normalized the increase in penile shaft collagen content , and the reduction in corporal smooth muscle cell ( SMC ) content , SMC/collagen , and replication index , and improved the lower collagen III/I ratio and the increase in apoptotic index , caused by P21554 , compared with sham operation . There were no effects of tadalafil on increased transforming growth factor beta1 , inducible nitric oxide synthase and xanthine oxidoreductase levels . CONCLUSIONS : A long-term single daily dose of tadalafil prevented CVOD and the underlying corporal fibrosis in the rat caused by CN damage , as effectively as the previously reported continuous treatment with vardenafil or sildenafil , through a cGMP-related mechanism that appears to be independent of inducible nitric oxide synthase induction . Potentiation of neurotoxicity in double-mutant mice with Pink1 ablation and A53T- P37840 overexpression . The common age-related neurodegeneration of Parkinson 's disease can result from dominant causes like increased dosage of vesicle-associated alpha-synuclein ( P37840 ) or recessive causes like deficiency of mitophagy factor Q9BXM7 . Interactions between these triggers and their convergence onto shared pathways are crucial , but currently conflicting evidence exists . Here , we crossed previously characterized mice with A53T- P37840 overexpression and with Pink1 deletion to generate double mutants ( DMs ) . We studied their lifespan and behavior , histological and molecular anomalies at late and early ages . DM animals showed potentiated phenotypes in comparison with both single mutants ( SMs ) , with reduced survival and strongly reduced spontaneous movements from the age of 3 months onwards . In contrast to SMs , a quarter of DM animals manifested progressive paralysis at ages > 1 year and exhibited protein aggregates immunopositive for pSer129- P37840 , p62 and ubiquitin in spinal cord and basal brain . Brain proteome quantifications of ubiquitination sites documented altered degradation of P37840 and the DNA-damage marker P16104 at the age of 18 months . Global brain transcriptome profiles and qPCR validation experiments identified many consistent transcriptional dysregulations already at the age of 6 weeks , which were absent from SMs . The observed downregulations for Dapk1 , Dcaf17 , Rab42 and the novel P37840 -marker Lect1 as well as the upregulations for Dctn5 , Mrpl9 , Tmem181a , Xaf1 and H2afx reflect changes in ubiquitination , mitochondrial/synaptic/microtubular/cell adhesion dynamics and DNA damage . Thus , our study confirmed that P37840 -triggered neurotoxicity is exacerbated by the absence of Q9BXM7 and identified a novel molecular signature that is detectable early in the course of this double pathology . P37840 A30P point-mutation generates age-dependent nigrostriatal deficiency in mice . Lewy bodies are mainly composed of alpha-synuclein ( P37840 ) and specific mutations in P37840 gene are related to familial forms of Parkinson 's disease ( PD ) . The purpose of our study was to generate a mouse line with A30P knock-in point mutation in P37840 gene and to test if a single point-mutation is able to turn otherwise normal P37840 into a toxic form . The behavioral profile of P37840 A30P mice was followed for 16 months . Generally , these mice are healthy and viable without any obvious abnormalities . Starting from the age of 13 months mice developed a significant deficit in motor performance tests related to nigrostriatal function ( ink-test and beam walk ) . In other tests ( motility boxes , rotarod ) mice continuously performed normally . Moreover , P37840 A30P mice expressed the altered sensitivity to Q05940 inhibitor reserpine , possibly reflecting a functional deficiency of dopamine . Indeed , mice at 15 months of age had significantly reduced levels of dopamine and its major metabolite DOPAC in the striatum , and reduced levels of dopamine in the mesolimbic system . The present study confirms that P37840 plays an important role in the development of PD and an insertion of a single point mutation is sufficient to generate age-related decline in specific motor performance . The generated mouse line has a potential to become a model for PD with comparable time course and phenotype . Glucocorticoid-induced surface expression of annexin 1 blocks beta2-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 ) . 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 . METHODS : Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate ( FP ) , and beta2-integrin adhesion was measured after stimulation with P05113 or eotaxin . Effects of FP on P47712 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 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 beta2-integrin adhesion . Translocation of P47712 to the nuclear membrane was significantly blocked by incubation with FP . Blockade was reversed with annexin 1 blocking antibody . CONCLUSION : Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1 , which blocks P47712 translocation to nuclear membrane . Growth-associated gene expression profiles by microarray analysis of trophoblast of molar pregnancies and normal villi . We used microarray analysis to investigate expression profiles of 589 known genes committed to cell growth control to characterize regulatory circuitry for cell proliferation in complete moles ( CMs ) . CMs are characterized by hyperplastic trophoblast and have a high propensity to give rise to choriocarcinoma . Characteristic alterations in gene expression profiles were observed when compared with normal villi . Fifty-seven genes were significantly up-regulated in CMs and involved the Ras-Map kinase 3 , Jak- P42229 , and Wnt signal pathways , implicating growth factor or cytokine-mediated signal pathways in the trophoblastic hyperplasia of CMs . Several genes associated with anti-apoptosis , cell structuring , and/or cell attachment were also up-regulated in CMs . In contrast , relatively fewer genes were down-regulated and these involved IGFBPs , versican , interleukin-1 , tumor necrosis factor receptor , P16070 , and P43351 . Genes identified in this study may elucidate regulation mechanisms of trophoblastic proliferation and mechanisms causing a pathological phenotype in CMs . Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries . BACKGROUND : Atherosclerosis affects aorta , coronary , carotid , and iliac arteries most frequently than any other body vessel . There may be common molecular pathways sustaining this process . Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis . RESULTS : We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression . We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis . Caveolae and JAK/ P35610 pathways , and P06702 / P05109 interacting proteins are certainly involved in the development of vascular disease . We found that the system of caveolae is directly connected with genes that respond to hormone receptors , and indirectly with the apoptosis pathway . Cytokines , chemokines and growth factors released in the blood flux were investigated in parallel . High levels of RANTES , IL-1ra , MIP-1 alpha , MIP-1 beta , P60568 , P05112 , P05113 , P05231 , P13232 , Q16552 , DB00102 , P15692 and P01579 were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels . CONCLUSION : The pattern of cytokine and P06702 / P05109 up-regulation characterizes atherosclerosis as a proinflammatory disorder . Activation of the JAK/ P35610 pathway is confirmed by the up-regulation of P05231 , P42224 , Q00978 and Q13651 genes in coronary and carotid plaques . The functional network constructed in our research is an evidence of the central role of P35610 protein and the caveolae system to contribute to preserve the plaque . Moreover , Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype . Inhibition of histamine H1 receptor activity modulates proinflammatory cytokine production of dendritic cells through c-Rel activity . BACKGROUND : DB11320 exerts diverse effects on immune regulation through four types of histamine receptors ( HRs ) . Among them , type 1 receptor ( P35367 ) plays an important role in allergic inflammation . Dendritic cells ( DCs ) , which express at least three types of HRs , are professional antigen-presenting cells controlling the development of allergic inflammation . However , the molecular mechanisms involved in P35367 -mediated NF-ĸB signaling of DCs remain poorly defined . METHODS : Bone-marrow ( BM ) -derived DCs ( BM-DCs ) were treated with P35367 inverse agonists to interrupt basal P35367 -mediated signaling . The crosstalk of P35367 -mediated signaling and the NF-ĸB pathway was examined by NF-ĸB cellular activity using a luciferase reporter assay , NF-ĸB subunit analysis using Western blotting and P01375 -α promoter activity using chromatin immunoprecipitation . RESULTS : Blockage of P35367 signaling by inverse agonists significantly inhibited P01375 -α and P05231 production of BM-DCs . P35367 -specific agonists were able to enhance P01375 -α production , but this overexpression was significantly inhibited by NF-ĸB inhibitor . The P35367 inverse agonist ketotifen also suppressed cellular NF-ĸB activity , suggesting crosstalk between P35367 and NF-ĸB signaling in DCs . After comprehensive analysis of NF-ĸB subunits , c-Rel protein expression was significantly down-regulated in ketotifen-treated BM-DCs , which led to inhibition of the promoter activity of P01375 -α . Finally , adoptive transfer of the ketotifen-treated BM-DCs did not induce significant allergic airway inflammation compared to that of control cells in vivo . CONCLUSIONS : Our results suggest that c-Rel controls P35367 -mediated proinflammatory cytokine production in DCs . This study provides a potential mechanism of P35367 -mediated signaling and NF-ĸB pathway crosstalk in allergic inflammation . Sources contributing to the average extracellular concentration of dopamine in the nucleus accumbens . Mesolimbic dopamine neurons fire in both tonic and phasic modes resulting in detectable extracellular levels of dopamine in the nucleus accumbens ( NAc ) . In the past , different techniques have targeted dopamine levels in the NAc to establish a basal concentration . In this study , we used in vivo fast scan cyclic voltammetry ( FSCV ) in the NAc of awake , freely moving rats . The experiments were primarily designed to capture changes in dopamine caused by phasic firing - that is , the measurement of dopamine ' transients ' . These FSCV measurements revealed for the first time that spontaneous dopamine transients constitute a major component of extracellular dopamine levels in the NAc . A series of experiments were designed to probe regulation of extracellular dopamine . DB00281 was infused into the ventral tegmental area , the site of dopamine cell bodies , to arrest neuronal firing . While there was virtually no instantaneous change in dopamine concentration , longer sampling revealed a decrease in dopamine transients and a time-averaged decrease in the extracellular level . Dopamine transporter inhibition using intravenous GBR12909 injections increased extracellular dopamine levels changing both frequency and size of dopamine transients in the NAc . To further unmask the mechanics governing extracellular dopamine levels we used intravenous injection of the vesicular monoamine transporter ( Q05940 ) inhibitor , tetrabenazine , to deplete dopamine storage and increase cytoplasmic dopamine in the nerve terminals . DB04844 almost abolished phasic dopamine release but increased extracellular dopamine to ∼500 nM , presumably by inducing reverse transport by dopamine transporter ( Q01959 ) . Taken together , data presented here show that average extracellular dopamine in the NAc is low ( 20-30 nM ) and largely arises from phasic dopamine transients . Molecular determinants of trastuzumab efficacy : What is their clinical relevance ? DB00072 -containing therapy is a standard of care for human epidermal growth factor receptor-2 ( P04626 ) -positive breast cancer . In pre-clinical models , a wide range of molecular mechanisms have been associated with reduced sensitivity to trastuzumab in vitro . These include expression of the truncated P04626 receptor fragment p95HER2 , activating mutation of the gene encoding the class 1A catalytic subunit of phosphatidylinositol 3-kinase ( P42336 ) , loss of phosphatase and tensin homolog ( P60484 ) , activation of other downstream signal transducers , prevention of cell cycle arrest , increased signaling through alternative ( HER or non-HER ) tyrosine kinase receptors , and resistance to antibody-dependent cellular cytotoxicity . However , the clinical significance of these mechanisms as determinants of trastuzumab efficacy in vivo has been unclear . Here , we review clinical studies of potential predictive biomarkers of trastuzumab efficacy in P04626 -positive breast cancer and consider whether evaluation of such markers might inform patient selection for therapy . We find that clinical evidence relating to potential predictive biomarkers is mostly limited to small , retrospective studies , many of which have yielded conflicting findings . Some trends are evident in the retrospective data and in biomarker analyses from randomized clinical trials , particularly relating to activation of the phosphatidylinositol 3-kinase pathway , but none is sufficiently strong to form a basis for patient selection . This may be explained by the fact that multiple mechanisms of action determine the clinical efficacy of trastuzumab . In the absence of novel , validated biomarkers of efficacy , trastuzumab eligibility should continue to be based on evaluation of P04626 status according to standard methods . Recombinant P17936 inhibits allergic lung inflammation , P15692 production , and vascular leak in a mouse model of asthma . BACKGROUND : Vascular endothelial growth factor ( P15692 ) plays a pro-inflammatory mediator as well as a vascular permeability factor in bronchial asthma . P01308 -like growth factor ( IGF ) -I is also involved in the inflammatory process associated with bronchial asthma and stimulates P15692 expression . The IGF-binding proteins ( IGFBPs ) , especially P17936 , display distinctive properties and can interfere with various biological processes . METHODS : In this study , an ovalbumin ( OVA ) -induced murine model of allergic airway disease was used to investigate which mechanism is implicated in the preventive and therapeutic actions of P17936 administered exogenously on allergen-induced bronchial inflammation and airway hyper-responsiveness , in particular focusing on the regulation of P15692 expression . RESULTS : Administration of recombinant human P17936 to OVA-inhaled mice substantially attenuated the increases in hypoxia-inducible factor ( HIF ) -α activity , P05019 production , and P15692 protein levels in the lung . In addition , the blockade of P05019 action decreased the OVA-induced P15692 expression , airway inflammation , and bronchial hyper-responsiveness . The administration of recombinant human P17936 or CBO-P11 also reduced significantly increases in inflammatory cells , airway hyper-responsiveness , levels of P05112 , P05113 , P35225 , and vascular permeability in the lung of OVA-inhaled mice . Moreover , when recombinant human P17936 was administered after the completion of OVA inhalation , these therapeutic effects of P17936 were also observed . CONCLUSIONS : These results indicate that P17936 administered exogenously may attenuate antigen-induced airway inflammation and hyper-responsiveness through the modulation of vascular leakage and P15692 expression mediated by HIF-1α/HIF-2α signaling as well as P05019 action in allergic airway disease of mice . P10275 accelerates premature senescence of human dermal papilla cells in association with DNA damage . The dermal papilla , located in the hair follicle , expresses androgen receptor and plays an important role in hair growth . Androgen/ P10275 actions have been implicated in the pathogenesis of androgenetic alopecia , but the exact mechanism is not well known . Recent studies suggest that balding dermal papilla cells exhibit premature senescence , upregulation of p16(INK4a) , and nuclear expression of DNA damage markers . To investigate whether androgen/AR signaling influences the premature senescence of dermal papilla cells , we first compared frontal scalp dermal papilla cells of androgenetic alopecia patients with matched normal controls and observed that premature senescence is more prominent in the dermal papilla cells of androgenetic alopecia patients . Exposure of androgen induced premature senescence in dermal papilla cells from non-balding frontal and transitional zone of balding scalp follicles but not in beard follicles . Overexpression of the AR promoted androgen-induced premature senescence in association with p16(INK4a) upregulation , whereas knockdown of the androgen receptor diminished the effects of androgen . An analysis of γ- P16104 expression in response to androgen/androgen receptor signaling suggested that DNA damage contributes to androgen/androgen receptor-accelerated premature senescence . These results define androgen/androgen receptor signaling as an accelerator of premature senescence in dermal papilla cells and suggest that the androgen/androgen receptor-mediated DNA damage-p16(INK4a) axis is a potential therapeutic target in the treatment of androgenetic alopecia . DB00107 alleviates the neuroendocrine and cytokine response to bacterial endotoxin in healthy men . DB00107 is a hormone and neurotransmitter found to have anti-inflammatory functions in rodents . Here we used experimental bacterial endotoxinemia to examine the role of exogenous oxytocin administration on innate immune responses in humans . Ten healthy men received , in a randomized , placebo-controlled , crossover design , placebo , oxytocin , LPS , and LPS + oxytocin . DB00107 treatment resulted in a transient or prolonged reduction of endotoxin-induced increases in plasma DB01285 , cortisol , procalcitonin , P01375 , IL-1 receptor antagonist , P05112 , P05231 , macrophage inflammatory protein-1alpha , macrophage inflammatory protein-1beta , monocyte chemoattractant protein-1 ( P13500 ) , interferon-inducible protein 10 , and P15692 . In vitro , oxytocin had no impact on LPS effects in releasing P01375 , P05231 , and P13500 in monocytes and peripheral blood mononuclear cells from healthy human donors . In summary , oxytocin decreases the neuroendocrine and cytokine activation caused by bacterial endotoxin in men , possibly due to the pharmacological modulation of the cholinergic anti-inflammatory pathway . DB00107 might be a candidate for the therapy of inflammatory diseases and conditions associated with high cytokine and P15692 levels . Antenatal maternally-administered phosphodiesterase type 5 inhibitors normalize P29474 expression in the fetal lamb model of congenital diaphragmatic hernia . PURPOSE : Pulmonary hypertension ( pHTN ) , a main determinant of survival in congenital diaphragmatic hernia ( Q8NE62 ) , results from in utero vascular remodeling . Phosphodiesterase type 5 ( O76074 ) inhibitors have never been used antenatally to treat pHTN . The purpose of this study is to determine if antenatal O76074 inhibitors can prevent pHTN in the fetal lamb model of Q8NE62 . METHODS : Q8NE62 was created in pregnant ewes . Postoperatively , pregnant ewes received oral placebo or tadalafil , a O76074 inhibitor , until delivery . Near term gestation , lambs underwent resuscitations , and lung tissue was snap frozen for protein analysis . RESULTS : Mean cGMP levels were 0.53±0.11 in placebo-treated fetal lambs and 1.73±0.21 in tadalafil-treated fetal lambs ( p=0.002 ) . Normalized expression of P29474 was 82 % ±12 % in Normal-Placebo , 61 % ±5 % in Q8NE62 -Placebo , 116 % ±6 % in Normal- DB00820 , and 86 % ±8 % in Q8NE62 - DB00820 lambs . Normalized expression of β-sGC was 105 % ±15 % in Normal-Placebo , 82 % ±3 % in Q8NE62 -Placebo , 158 % ±16 % in Normal- DB00820 , and 86 % ±8 % in Q8NE62 - DB00820 lambs . P29474 and β-sGC were significantly decreased in Q8NE62 ( p=0.0007 and 0.01 for P29474 and β-sGC , respectively ) , and tadalafil significantly increased P29474 expression ( p=0.0002 ) . CONCLUSIONS : O76074 inhibitors can cross the placental barrier . β-sGC and P29474 are downregulated in fetal lambs with Q8NE62 . Antenatal O76074 inhibitors normalize P29474 and may prevent in utero vascular remodeling in Q8NE62 . Regulation of double-strand break-induced mammalian homologous recombination by P63165 , a Q96B01 . Mammalian Q06609 protein plays essential roles in DNA homologous recombination , DNA repair and cell proliferation . Q06609 activities are regulated by its associated proteins . It was previously reported that a ubiquitin-like protein , P63165 , associates with Q06609 in the yeast two-hybrid system . One function of P63165 is to covalently conjugate with target proteins and thus modify their function . In the present study we found that non-conjugated P63165 forms a complex with Q06609 and P43351 proteins in human cells . Overexpression of P63165 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells . With or without overexpressed P63165 , most homologous recombination products arise by gene conversion . However , overexpression of P63165 reduces the fraction of bidirectional gene conversion tracts . Overexpression of a mutant P63165 that is incapable of being conjugated retains the ability to inhibit homologous recombination . These results suggest a regulatory role for P63165 in homologous recombination . DB00184 induces cell proliferation , invasion and epithelial-mesenchymal transition in a variety of human cancer cell lines . Cigarette smoking is strongly correlated with the onset of nonsmall cell lung cancer ( NSCLC ) . DB00184 , an active component of cigarettes , has been found to induce proliferation of lung cancer cell lines . In addition , nicotine can induce angiogenesis and confer resistance to apoptosis . All these events are mediated through the nicotinic acetylcholine receptors ( nAChRs ) on lung cancer cells . In this study , we demonstrate that nicotine can promote anchorage-independent growth in NSCLCs . In addition , nicotine also induces morphological changes characteristic of a migratory , invasive phenotype in NSCLCs on collagen gel . These morphological changes were similar to those induced by the promigratory growth factor P15692 . The proinvasive effects of nicotine were mediated by alpha7-nAChRs on NSCLCs . RT-PCR analysis showed that the alpha7-nAChRs were also expressed on human breast cancer and pancreatic cancer cell lines . DB00184 was found to promote proliferation and invasion in human breast cancer . The proinvasive effects of nicotine were mediated via a nAChR , Src and calcium-dependent signaling pathway in breast cancer cells . In a similar fashion , nicotine could also induce proliferation and invasion of Aspc1 pancreatic cancer cells . Most importantly , nicotine could induce changes in gene expression consistent with epithelial to mesenchymal transition ( EMT ) , characterized by reduction of epithelial markers like P12830 expression , ZO-1 staining and concomitant increase in levels of mesenchymal proteins like vimentin and fibronectin in human breast and lung cancer cells . Therefore , it is probable that the ability of nicotine to induce invasion and EMT may contribute to the progression of breast and lung cancers . Topoisomerase II-mediated DNA cleavage and mutagenesis activated by nitric oxide underlie the inflammation-associated tumorigenesis . AIMS : Both cancer-suppressing and cancer-promoting properties of reactive nitrogen and oxygen species ( RNOS ) have been suggested to play a role in tumor pathology , particularly those activities associated with chronic inflammation . Here , we address the impact of nitric oxide ( NO ) on the induction of DNA damage and genome instability with a specific focus on the involvement of topoisomerase II ( P11388 ) . We also investigate the contribution of NO to the formation of skin melanoma in mice . RESULTS : Similar to the P11388 -targeting drug , etoposide ( DB00773 ) , the NO-donor , S-nitrosoglutathione ( GSNO ) , induces skin melanomas formation in 7,12-dimethyl- benz[a]anthracene ( DMBA ) -initiated mice . To explore the mechanism(s) underlying this NO-induced tumorigenesis , we use a co-culture model system to demonstrate that inflamed macrophages with inducible NO synthase ( P35228 ) expression cause γ- P16104 activation , p53 phosphorylation , and chromosome DNA breaks in the target cells . Inhibitor experiments revealed that NO and P11388 isozymes are responsible for the above described cellular phenotypes . Notably , NO , unlike DB00773 , preferentially induces the formation of TOP2β cleavable complexes ( TOP2βcc ) in cells . Moreover , GSNO induced P11388 -dependent DNA sequence rearrangements and cytotoxicity . Furthermore , the incidences of GSNO- and DB00773 -induced skin melanomas were also observed to be lower in the skin-specific top2β-knockout mice . Our results suggest that P11388 isozymes contribute to NO-induced mutagenesis and subsequent cancer development during chronic inflammation . INNOVATION AND CONCLUSIONS : We provide the first experimental evidence for the functional role of P11388 in NO-caused DNA damage , mutagenesis , and carcinogenesis . Notably , these studies contribute to our molecular understanding of the cancer-promoting actions of RNOS during chronic inflammation . Changing paradigms in management of metastatic Castration Resistant Prostate Cancer ( mCRPC ) . Recently , the standard of care for metastatic Castration Resistant Prostate Cancer ( mCRPC ) has changed considerably . Persistent androgen receptor ( AR ) signaling has been identified as a target for novel therapies and reengages the fact that AR continues to be the primary target responsible for metastatic prostate cancer . P10275 gene amplification and over expression have been found to result in a higher concentration of androgen receptors on tumor cells , making them extremely sensitive to low levels of circulating androgens . Additionally , prostate cancer cells are able to maintain dihydrotestosterone ( DB02901 ) concentration in excess of serum concentrations to support tumor growth . For many years ketoconazole was the only P05093 inhibitor that was used to treat mCRPC . However , significant toxicities limit its use . Newly approved chemotherapeutic agents such as DB05812 ( an oral selective inhibitor of CYP17A ) , which blocks androgen biosynthesis both within and outside the prostate cancer cells ) , and enzalutamide ( blocks AR signaling ) have improved overall survival . There are also ongoing phase III trials for Orteronel ( P50750 - 700 ) , ARN- 509 and Galeterone ( TOK-001 ) , which targets androgen signaling . In this review , we will present the rationale for the newly approved hormonal treatments , their indications and complications , and we will discuss ongoing trials that are being done to improve the efficacy of the approved agents . Finally , we will talk about the potential upcoming hormonal treatments for mCRPC . Sporadic breast carcinomas with somatic P38398 gene deletions share genotype/phenotype features with familial breast carcinomas . BACKGROUND : High frequencies of loss of heterozygosity ( LOH ) are found in familial breast carcinomas with BRCA mutations . Although LOH of P38398 does not coincide with somatic P38398 mutations , reduced P38398 protein expression and hypermethylation indicate the involvement of P38398 in sporadic carcinogenesis . To further investigate the role of BRCA we determined LOH of P38398 and correlated this with LOH in other breast cancer-associated regions . MATERIALS AND METHODS : A total of 105 sporadic breast carcinomas were analysed for LOH in the regions of P38398 , P51587 , P04637 , Caveolin1 , " putative BRCA3 " , P60484 , Q13315 and P12830 and correlated it with clinicopathological features . RESULTS : We found an overall increase of LOH in carcinomas with simultaneous LOH of P38398 . Significantly higher LOH rates were detected in the regions of P04637 ( 80 % : 34.7 % ; p < 0.005 ) , 8q21 ( 72.7 % : 30.6 % ; p < 0.010 ) and 10q22-23 ( 21.1 % : 5.9 % ; p=0.043 ) . Moreover , estrogen receptor-negative carcinomas revealed LOH of P38398 more frequently than estrogen receptor-positive carcinomas ( 39 % : 12 % ; p=0.003 ) . CONCLUSION : These data indicate that LOH of P38398 coincides with a defect of the DNA repair pathway . Therefore , LOH of P38398 determines a subgroup of sporadic breast carcinomas sharing genotype/phenotype features with familial breast carcinomas . Q00987 is a ubiquitin ligase of P12956 -Akt promotes cell survival by inhibiting Q00987 -dependent P12956 destabilization . Earlier , we have reported that 70 kDa subunit of Ku protein heterodimer ( P12956 ) binds and inhibits Bax activity in the cytosol and that ubiquitin ( Ub ) -dependent proteolysis of cytosolic P12956 facilitates Bax-mediated apoptosis . We found that Q00987 ( human homolog of murine double minute ) has an ability to ubiquitinate P12956 and that Q00987 overexpression in cultured cells causes a decrease in P12956 expression levels . An interaction between P12956 and Q00987 was shown by means of immunoprecipitation , whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Q00987 . Vascular endothelial growth factor ( P15692 ) is known to inhibit endothelial cell ( EC ) apoptosis through an Akt-mediated survival kinase signal ; however , the mechanism underlying this inhibition of apoptosis has not been fully elucidated . We found that P15692 inhibited cytosolic P12956 degradation induced by apoptotic stress . It is known that Akt-dependent phosphorylation of Q00987 causes nuclear translocation of Q00987 followed by Q00987 -mediated inactivation of p53 . We found that P15692 stimulated nuclear translocation of Q00987 in EC and efficiently inhibited P12956 degradation . We also found that constitutively active Akt , but not kinase-dead Akt , inhibited P12956 degradation in the cytosol . Furthermore , P12956 knockdown diminished antiapoptotic activity of Akt . Taken together , we propose that Q00987 is a P12956 Ub ligase and that Akt inhibits Bax-mediated apoptosis , at least in part , by maintaining P12956 levels through the promotion of Q00987 nuclear translocation . Loss of homologous recombination or non-homologous end-joining leads to radial formation following DNA interstrand crosslink damage . High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination . The mechanism of radial formation observed following DNA damage has yet to be determined . Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway , we speculated that radials might be the result of defects in either of the pathways of DNA repair . To test this hypothesis , we have investigated the role of homologous recombination proteins Q06609 and P43351 , non-homologous end-joining proteins P12956 and P49917 , and protein P49959 in radial formation and cell survival following interstrand crosslink damage with mitomycin C . For the studies we used small inhibitory RNA to deplete the proteins from cells , allowing for evaluation of radial formation and cell survival . In transformed normal human fibroblasts , depletion of these proteins increased interstrand crosslink sensitivity as manifested by decreased cell survival and increased radial formation . These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks , indicating each pathway plays a role in the normal response to interstrand crosslink damage . We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation , since radials occur with depletion of these pathways . Identification of an acetylation-dependant P12956 /FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis . FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors . We report a novel interaction between FLIP and the DNA repair protein P12956 that regulates FLIP protein stability by inhibiting its polyubiquitination . Furthermore , we found that the histone deacetylase ( HDAC ) inhibitor DB02546 ( DB02546 ) enhances the acetylation of P12956 , thereby disrupting the FLIP/ P12956 complex and triggering FLIP polyubiquitination and degradation by the proteasome . Using in vitro and in vivo colorectal cancer models , we further demonstrated that DB02546 -induced apoptosis is dependant on FLIP downregulation and caspase 8 activation . In addition , an Q9UBN7 -specific inhibitor Tubacin recapitulated the effects of DB02546 , suggesting that Q9UBN7 is a key regulator of P12956 acetylation and FLIP protein stability . Thus , HDAC inhibitors with anti- Q9UBN7 activity act as efficient post-transcriptional suppressors of FLIP expression and may , therefore , effectively act as ' FLIP inhibitors ' . Mitoxantrone inhibits HIF-1α expression in a topoisomerase II-independent pathway . PURPOSE : Solid tumors encounter a growth-limiting hypoxic microenvironment as they develop . Hypoxia-inducible factors ( HIF ) play important roles in hypoxia-associated tumor development and therapeutic resistance . Targeting the HIF pathway ( especially HIF-1α ) represents a promising cancer treatment strategy . Here , we report a novel class of HIF-1α inhibitors and the possible molecular basis of inhibition . EXPERIMENTAL DESIGN : We analyzed the inhibitory effects of clinically used topoisomerase II ( P11388 ) -targeting drugs on HIF-1α expression with a primary focus on mitoxantrone . The potential role of P11388 in mitoxantrone-inhibited HIF-1α expression was studied using pharmacologic inhibition , a knockdown approach , and P11388 mutant cells . Moreover , involvement of mitoxantrone in proteasome-mediated degradation , transcription , and translation of HIF-1α was examined . RESULTS : The P11388 -targeting mitoxantrone , but neither doxorubicin nor etoposide ( DB00773 ) , strongly inhibited HIF-1α expression under hypoxic conditions in a dose- and time-dependent manner . Surprisingly , the mitoxantrone-mediated inhibition of HIF-1α expression was largely independent of two P11388 isozymes , proteasomal degradation , and transcription . Furthermore , mitoxantrone inhibited HIF-1α expression and function in a similar fashion as cycloheximide , suggesting that mitoxantrone might inhibit HIF-1α via a blockage at its translation step . In vitro translation experiments using HIF-1α mRNA further confirmed inhibition of HIF-1α translation by mitoxantrone . Interestingly , levels of the polysome-bound HIF-1α and P15692 mRNA were elevated and decreased after mitoxantrone treatment , respectively . CONCLUSIONS : We have identified the P11388 -targeting compound , mitoxantrone , as an HIF-1α inhibitor possibly through a translation inhibition mechanism , suggesting the possibility of an additional anticancer activity for mitoxantrone . Blockade of cannabinoid receptors reduces inflammation , leukocyte accumulation and neovascularization in a model of sponge-induced inflammatory angiogenesis . OBJECTIVE : Angiogenesis depends on a complex interaction between cellular networks and mediators . The endocannabinoid system and its receptors have been shown to play a role in models of inflammation . Here , we investigated whether blockade of cannabinoid receptors may interfere with inflammatory angiogenesis . MATERIALS AND METHODS : Polyester-polyurethane sponges were implanted in C57Bl/6j mice . Animals received doses ( 3 and 10 mg/kg/daily , s.c. ) of the cannabinoid receptor antagonists SR141716A ( P21554 ) or SR144528 ( CB2 ) . Implants were collected at days 7 and 14 for cytokines , hemoglobin , myeloperoxidase , and N-acetylglucosaminidase measurements , as indices of inflammation , angiogenesis , neutrophil and macrophage accumulation , respectively . Histological and morphometric analysis were also performed . RESULTS : Cannabinoid receptors expression in implants was detected from day 4 after implantation . Treatment with P21554 or CB2 receptor antagonists reduced cellular influx into sponges at days 7 and 14 after implantation , although P21554 receptor antagonist were more effective at blocking leukocyte accumulation . There was a reduction in P01375 -α , P15692 , P09341 /KC , P13500 /JE , and P10147 /MIP-1α levels , with increase in P13501 /RANTES . Both treatments reduced neovascularization . Dual blockade of cannabinoid receptors resulted in maximum inhibition of inflammatory angiogenesis . CONCLUSIONS : Blockade of cannabinoid receptors reduced leukocyte accumulation , inflammation and neovascularization , suggesting an important role of endocannabinoids in sponge-induced inflammatory angiogenesis both via P21554 and CB2 receptors . DB00072 has opposing effects on SN-38-induced double-strand breaks and cytotoxicity in P04626 -positive gastric cancer cells depending on administration sequence . AIM : We investigated the effects of trastuzumab , an anti- P04626 humanized monoclonal antibody , on DNA breaks induced by SN-38 , a topoisomerase-1 inhibitor , in gastric cancer cell lines positive or negative for P04626 expression . MATERIALS AND METHODS : NCI-N87 ( P04626 + ) and MKN74 ( P04626 - ) cells were exposed to SN-38 in the presence or absence of trastuzumab . DB00072 was added either prior to or after SN-38 . Effects of trastuzumab on the induction of gamma- P16104 , a marker of DNA double-strand breaks , the cytotoxicity of SN-38 and cell cycle progression were determined . RESULTS : When trastuzumab was administered following SN-38 , it increased γ P16104 levels and cytotoxicity of SN-38 in NCI-N87 cells , but not in MKN74 cells . In contrast , pretreatment with trastuzumab reduced SN-38-induced γ P16104 expression and cytotoxicity of SN-38 in NCI-N87 cells , but not in MKN74 cells . DB00072 delayed cell cycle progression in NCI-N87 cells only . CONCLUSION : DB00072 has opposing effects on SN-38-induced double-strand breaks and cytotoxicity depending on the order of administration of the two agents . P35367 antagonist cetirizine impairs working memory processing speed , but not episodic memory . BACKGROUND AND PURPOSE : The histaminergic neurotransmitter system is currently under investigation as a target for drug treatment of cognitive deficits in clinical disorders . The therapeutic potential of new drugs may initially be screened using a model of histaminergic dysfunction , for example , as associated with the use of centrally active antihistamines . Of the selective second generation antihistamines , cetirizine has been found to have central nervous system effects . The aim of the present study was to determine whether cetirizine can be used as a tool to model cognitive deficits associated with histaminergic hypofunction . EXPERIMENTAL APPROACH : The study was conducted according to a three-way , double-blind , cross-over design . Treatments were single oral doses of cetirizine 10 and 20 mg and placebo . Effects on cognition were assessed using tests of word learning , memory scanning , vigilance , divided attention , tracking and visual information processing speed . KEY RESULTS : DB00341 10 mg impaired tracking performance and both doses impaired memory scanning speed . None of the other measures indicated impaired performance . CONCLUSION AND IMPLICATIONS : DB00341 affects information processing speed , but these effects were not sufficient to serve as a model for cognitive deficits in clinical disorders . Guggulsterone inhibits angiogenesis by blocking P40763 and P15692 expression in colon cancer cells . The plant sterol guggulsterone has been shown to exert anti-tumor effects , making it a candidate chemotherapeutic agent . We investigated the anti-tumor effects of guggulsterone on colon cancer cells and elucidated the underlying molecular mechanisms related to angiogenesis . The apoptotic effects of guggulsterone were examined by cell survival assay . Western blot analysis was used to determine the levels of various down-stream intracellular proteins involved in angiogenesis , including signal transducer and activator of transcription 3 ( P40763 ) , vascular endothelial growth factor ( P15692 ) , hypoxia-inducible factor-1alpha ( HIF-1alpha ) and aryl hydrocarbon receptor nuclear translocator ( P27540 ) . Using chromatin immunoprecipitation assay , we tested whether guggulsterone affects the recruitment of P40763 , P27540 and HIF-1alpha to the human P15692 promoter . To investigate the effect of guggulsterone on vascular endothelial cell migration and invasion , tube formation and migration assays were conducted using human umbilical vein endothelial cells ( HUVECs ) . Matrix metalloproteinase ( MMP ) -2 and -9 activities were measured by gelatin zymography . Guggulsterone significantly reduced cell viability in colon cancer cells in a dose-dependent manner and blocked P15692 , P27540 and P40763 expression prominently in hypoxic conditions . The recruitment of P40763 and P27540 , but not HIF-1alpha , to the P15692 promoter was inhibited by guggulsterone treatment . HUVECs produced much foreshortened and severely broken tubes and showed decreased migration activity under guggulsterone effects . In addition , zymography revealed that P08253 and -9 enzyme activities were markedly lower in the presence of guggulsterone . The results of this study suggest that guggulsterone not only induces apoptosis , but also inhibits angiogenesis and metastasis in colon cancer cells by blocking P40763 and P15692 expression , suggesting its therapeutic potential in the treatment of colorectal cancer . P10275 YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration . X-linked spinal and bulbar muscular atrophy ( SBMA ) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration . SBMA is caused by polyglutamine repeat expansions in the androgen receptor ( AR ) . To determine the basis of AR polyglutamine neurotoxicity , we introduced human AR yeast artificial chromosomes carrying either 20 or 100 CAGs into mouse embryonic stem cells . The AR100 transgenic mice developed a late-onset , gradually progressive neuromuscular phenotype accompanied by motor neuron degeneration , indicating striking recapitulation of the human disease . We then tested the hypothesis that polyglutamine-expanded AR interferes with CREB binding protein ( CBP ) -mediated transcription of vascular endothelial growth factor ( P15692 ) and observed altered CBP-AR binding and P15692 reduction in AR100 mice . We found that mutant AR-induced death of motor neuron-like cells could be rescued by P15692 . Our results suggest that SBMA motor neuronopathy involves altered expression of P15692 , consistent with a role for P15692 as a neurotrophic/survival factor in motor neuron disease . Role of nitrative and oxidative DNA damage in inflammation-related carcinogenesis . Chronic inflammation induced by biological , chemical , and physical factors has been found to be associated with the increased risk of cancer in various organs . We revealed that infectious agents including liver fluke , Helicobacter pylori , and human papilloma virus and noninfectious agents such as asbestos fiber induced P35228 -dependent formation of 8-nitroguanine and 8-oxo-7 , 8-dihydro-2'-deoxyguanosine ( 8-oxodG ) in cancer tissues and precancerous regions . Our results with the colocalization of phosphorylated Q13315 and γ- P16104 with 8-oxodG and 8-nitroguanine in inflammation-related cancer tissues suggest that DNA base damage leads to double-stranded breaks . It is interesting from the aspect of genetic instability . We also demonstrated P05231 -modulated P35228 expression via P40763 and P00533 in Epstein-Barr-virus-associated nasopharyngeal carcinoma and found promoter hypermethylation in several tumor suppressor genes . Such epigenetic alteration may occur by controlling the DNA methylation through P05231 -mediated JAK/ P40763 pathways . Collectively , 8-nitroguanine would be a useful biomarker for predicting the risk of inflammation-related cancers . Gender difference in the activity but not expression of estrogen receptors alpha and beta in human lung adenocarcinoma cells . The higher frequency of lung adenocarcinoma in women smokers than in men smokers suggests a role for gender-dependent factors in the etiology of lung cancer . We evaluated estrogen receptor ( ER ) alpha and beta expression and activity in human lung adenocarcinoma cell lines and normal lung fibroblasts . Q8N1N2 -length ERalpha and ERbeta proteins were expressed in all cell lines with higher ERbeta than ERalpha . Although estradiol ( E(2) ) binding was similar , E(2) stimulated proliferation only in cells from females , and this response was inhibited by anti-estrogens 4-hydroxytamoxifen ( DB04468 ) and DB00947 . In contrast , E(2) did not stimulate replication of lung adenocarcinoma cells from males and DB04468 or ICI did not block cell proliferation . Similarly , transcription of an estrogen response element-driven reporter gene was stimulated by E(2) in lung adenocarcinoma cells from females , but not males . P06401 ( PR ) expression was increased by E(2) in two out of five adenocarcinoma cell lines from females , but none from males . E(2) decreased P12830 protein expression in some of the cell lines from females , as it did in MCF-7 breast cancer cells , but not in the cell lines from males . Thus , ERalpha and ERbeta expression does not correlate with the effect of ER ligands on cellular activities in lung adenocarcinoma cells . On the other hand , coactivator Q15648 expression was higher in lung adenocarcinoma cells from females versus males and higher in adenocarcinoma cells than in normal human bronchial epithelial cells . Q15648 and other ER coregulators may contribute to differences in estrogen responsiveness between lung adenocarcinoma cells in females and males . 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside ameliorates vascular senescence and improves blood flow involving a mechanism of p53 deacetylation . BACKGROUND AND AIMS : 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside ( THSG ) , a resveratrol analog with glucoside , has been shown in various studies to inhibit proliferation of vascular smooth muscle cells , attenuate inflammation , and prevent vascular endothelial dysfunction . In the study , we examined the effects of THSG on vascular senescence and blood flow . METHODS AND RESULTS : Oral administration of THSG for 14 weeks , resulted in notable increases in blood flow in spontaneously hypertensive rats ( SHRs ) ; and effective inhibition of vascular senescence as indicated by senescence-associated β-galactosidase ( SA-β-gal ) staining , phosphorylation of γ P16104 observed by stain analysis of immunofluorescence , and K373 acetylation of p53 in the aortic arches of SHRs . Oral administration of THSG also induced P29474 expression and urinary NOx production . THSG weekly activated Q96EB6 activity , stimulated P29474 promoter reporter gene activity , and ameliorated H(2)O(2)-induced cellular senescence and K373 acetylation of p53 in cultured human umbilical vein endothelial cells ( HUVECs ) . CONCLUSIONS : THSG improves blood flow and ameliorates vascular senescence by increasing P29474 expression and Sirt1 activity and decreasing acetylation of p53 at K373 site , at least in part , both in vitro and in vivo . Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II . Increasing the cellular concentration of DNA topoisomerase II in yeast by expressing constitutively a plasmid-borne P11388 gene encoding the enzyme greatly increases the sensitivity of the cells to amsacrine and etoposide ( DB00773 ) . This increased drug sensitivity at a higher intracellular DNA topoisomerase II level is observed in both P43351 + repair-proficient strains and rad52 mutants that are defective in the repair of double-stranded breaks . These results provide strong support of the hypothesis that the cellular target of these drugs is DNA topoisomerase II , and that these drugs kill cells by converting DNA topoisomerase II into a DNA damaging agent . Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation : attenuation of Q9Y6W8 , P05112 , and Q9HBE4 , but not P10747 , P13232 , and P40933 responses . The program death 1 ( P18621 ) receptor and its ligands , P18621 ligand (PD-L)1 and Q9BQ51 , define a novel regulatory pathway with potential inhibitory effects on T , B , and monocyte responses . In the present study , we show that human P01730 (+) T cells express P18621 , Q9NZQ7 , and Q9BQ51 upon activation , and Abs to the receptor can be agonists or antagonists of the pathway . Under optimal conditions of stimulation , Q9Y6W8 but not P10747 costimulation can be prevented by P18621 engagement . P60568 levels induced by costimulation are critical in determining the outcome of the P18621 engagement . Thus , low to marginal P60568 levels produced upon Q9Y6W8 costimulation account for the greater sensitivity of this pathway to P18621 -mediated inhibition . Interestingly , exogenous P60568 , P13232 , and P40933 but not P05112 and Q9HBE4 can rescue P18621 inhibition , suggesting that among these cytokines only those that activate P42229 can rescue P18621 inhibition . As P42229 has been implicated in the maintenance of IL-2Ralpha expression , these results suggest that P13232 and P40933 restore proliferation under conditions of P18621 engagement by enhancing high-affinity IL-2R expression and hence , P60568 responsiveness . Regulation of microphthalmia-associated transcription factor O75030 protein levels by association with the ubiquitin-conjugating enzyme hUBC9 . The basic helix-loop-helix/leucine zipper ( bHLH/ Q8N5A5 ) microphthalmia-associated transcription factor ( O75030 ) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells . To determine how O75030 activity is regulated , we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with O75030 . The majority of clones that showed positive interaction with a 158-amino-acid region of O75030 containing the bHLH/ Q8N5A5 domain ( aa 168-325 ) encoded the ubiquitin conjugating enzyme hUBC9 . The association of O75030 with hUBC9 was further confirmed by an in vitro Q86UG4 pull-down assay . Although hUBC9 is known to interact preferentially with SENTRIN/ P63165 , in vitro transcription/translation analysis demonstrated greater association of O75030 with ubiquitin than with SENTRIN . Importantly , cotransfection of O75030 and hUBC9 expression vectors resulted in O75030 protein degradation . O75030 protein was stabilized by the proteasome inhibitor MG132 , indicating the role of the ubiquitin-proteasome system in O75030 degradation . DB00133 73 , which is located in a region rich in proline , glutamic acid , serine , and threonine ( PEST ) , regulates O75030 protein stability , since a serine to alanine mutation prevented hUBC9-mediated O75030 ( S73A ) degradation . Furthermore , we identified lysine 201 as a potential ubiquitination site . A lysine to arginine mutation abolished O75030 ( K201R ) degradation by hUBC9 in vivo . Our experiments indicate that by targeting O75030 for proteasome degradation , hUBC9 is a critical regulator of melanocyte differentiation . P60484 sumo-wrestles human P43351 to mystery land . DB00588 -induced regulation of the balance within macrophage subpopulations . In asthma , treatment with inhaled corticosteroids reduces chronic peribronchial inflammation and restores the balance within macrophage subpopulations . This study investigates whether corticosteroids can regulate monocyte differentiation in vitro and thereby influence the balance of functionally distinct macrophages . Graded doses of fluticasone propionate ( FP ) were added to cultures of normal peripheral blood monocytes in the presence or absence of P05112 . Cells were harvested after 7 days ' culture . Double immunofluorescence studies were performed on cytospins of differentiated macrophages using the MoAbs RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes . Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring tumour necrosis factor-alpha ( P01375 ) production . FP reduced the number of mature cells with a D1+ antigen-presenting phenotype and up-regulated the development of cells with the D1/D7+ and D7+ phenotypes . Functionally , this was associated with reduced stimulation of T cell proliferation in a mixed leucocyte reaction ( P08235 ) . DB00588 also reversed the increase in both D1+ expression and P01375 production induced by P05112 . The effect of FP persisted for 24 h after removal of FP from the culture medium . These results suggest that FP treatment of asthmatics may have a direct beneficial effect by normalizing the macrophage subset imbalance that contributes to the chronic peribronchial inflammation present in this condition . New perspectives of vesicular monoamine transporter 2 chemical characteristics in mammals and its constant expression in type 1 diabetes rat models . Vesicular monoamine transporter 2 ( Q05940 ) has been exploited as a biomarker of β-cell mass in human islets . However , a current report suggested no immunoreactivity of Q05940 in the β cells of rat islets . To investigate the cellular localization of Q05940 in islets further , the pancreatic tissues from monkeys and humans were compared with those of rats and mice . The study was performed using among-species comparisons and a type 1 diabetes model ( T1DM ) for rats by Western blotting , double-label immunofluorescence , and confocal laser scanning microscopy . We found that Q05940 -immunoreactivity ( IR ) was distributed peripherally in the islets of rodents , but was widely scattered throughout the islets of primates . Consistent with rodent islets , Q05940 -IR did not exist in insulin ( P01308 ) -IR cells but was abundantly present in glucagon ( GLU ) -IR and pancreatic polypeptide ( PP ) -IR cells in monkey and human islets . Q05940 -IR had no colocalization with P01308 -IR in any part of the rat pancreas ( head , body , and tail ) . P01308 -IR cells were reduced dramatically in T1DM rat islets , but no significant alteration in the proportion of Q05940 -IR cells and GLU-IR cells was observed . Furthermore , a strong colocalization of Q05940 -IR with GLU-IR was distributed in the peripheral regions of diabetic islets . For the first time , the current study demonstrates the presence of Q05940 in α cells and PP cells but not in β cells in the islets of monkeys and humans . This study provides convinced morphologic evidence that Q05940 is not present in β cells . There needs to be studies for new markers for β cell mass . Selective inhibition of histone deacetylase 6 ( Q9UBN7 ) induces DNA damage and sensitizes transformed cells to anticancer agents . Q9UBN7 ( Q9UBN7 ) is structurally and functionally unique among the 11 human zinc-dependent histone deacetylases . Here we show that chemical inhibition with the Q9UBN7 -selective inhibitor tubacin significantly enhances cell death induced by the topoisomerase II inhibitors etoposide and doxorubicin and the pan-HDAC inhibitor DB02546 ( vorinostat ) in transformed cells ( LNCaP , MCF-7 ) , an effect not observed in normal cells ( human foreskin fibroblast cells ) . The inactive analogue of tubacin , nil-tubacin , does not sensitize transformed cells to these anticancer agents . Further , we show that down-regulation of Q9UBN7 expression by shRNA in LNCaP cells enhances cell death induced by etoposide , doxorubicin , and DB02546 . Tubacin in combination with DB02546 or etoposide is more potent than either drug alone in activating the intrinsic apoptotic pathway in transformed cells , as evidenced by an increase in PARP cleavage and partial inhibition of this effect by the pan-caspase inhibitor Z-VAD-fmk . Q9UBN7 inhibition with tubacin induces the accumulation of γ P16104 , an early marker of DNA double-strand breaks . Tubacin enhances DNA damage induced by etoposide or DB02546 as indicated by increased accumulation of γ P16104 and activation of the checkpoint kinase Chk2 . Tubacin induces the expression of P35638 ( P35638 / P35638 ) , a transcription factor up-regulated in response to cellular stress . P35638 induction is further increased when tubacin is combined with DB02546 . These findings point to mechanisms by which Q9UBN7 -selective inhibition can enhance the efficacy of certain anti-cancer agents in transformed cells . Prolonged treatment with bicalutamide induces androgen receptor overexpression and androgen hypersensitivity . BACKGROUND : Various hormone refractory prostate cancer cell models have been established with androgen depletion and have helped to clarify the mechanism for the transition into androgen-depletion independent status . However , the mechanism of bicalutamide resistance remains unclear because few cell models have been generated . METHODS : We generated a bicalutamide-resistant subline , LNCaP- O43633 , from LNCaP after prolonged treatment with bicalutamide . Androgen and/or bicalutamide responsiveness for proliferation and prostate-specific antigen ( PSA ) secretion were examined in vitro and in vivo . DB00624 and dihydrotestosterone ( DB02901 ) levels in xenografted tumors were analyzed by liquid chromatography-tandem mass spectrometry . P10275 ( AR ) gene mutation and amplification and AR and pAR(210) expression were determined . RESULTS : LNCaP- O43633 did not grow in an androgen-depleted medium and proliferation was stimulated in a tenfold lower concentration of androgen than that of LNCaP . LNCaP- O43633 grew in castrated male mice , and the DB02901 level in grafted LNCaP- O43633 tumors was 7.7-fold lower than in LNCaP tumors . DB01128 stimulated LNCaP- O43633 proliferation and PSA secretion in vitro and the antitumor activity of bicalutamide against LNCaP- O43633 was weaker than that of LNCaP in vivo . Additional AR mutation and AR gene amplification were not detected in LNCaP- O43633 , but AR and pAR(210) expression and PSA secretion in LNCaP- O43633 were higher than in LNCaP . CONCLUSIONS : DB01128 -resistant LNCaP- O43633 exhibited AR overexpression and hypersensitivity to low levels of androgen . Our data suggests that AR overexpression is a significant mechanism of bicalutamide resistance similar to resistance from chronic androgen depletion . In addition , pAR(210) overexpression could be a potential mechanism for hypersensitivity to low androgen in LNCaP- O43633 . Current researches on breast cancer epidemiology in Korea . As a cause of death in women , breast cancer ranks second to stomach cancer in Korea . Age-standardized mortality rates for breast cancer steadily increased during the 1980s and 1990s . There are big differences in the incidence rates for breast cancer compared with Western countries . Epidemiological features , trends in morbidity and mortality , various age-specific incidence curves , migrant study results , and analysis of the risk factors , however , suggest that the incidence of breast cancer might be further increasing in Korea . The key epidemiological hormonal risk factors for breast cancer are all explicable in terms of the estrogen augmented by progesterone hypothesis . These include older age , family history of breast cancer , early menarche , late menopause , late full-term pregnancy , and never a breast feeding . Both the establishment of high-risk groups and the estimation of lifetime risk are essential to develop a control strategy against breast cancer . Invasive ductal carcinoma is the most common histologic type of breast cancer in Korea , and the five-year survival rate has been estimated as 80-83 % . Recent studies on the identification of susceptibility factors such as genetic polymorphisms of P09488 /T1/P1 , P21964 , P05181 , P11511 , P05093 , P03372 , P18887 , O43542 , P43351 , TGF-alpha , P01375 , IL-1B , IL-1RN , P50613 etc. that predispose individuals to breast cancer by gene-environment or gene-gene interactions may possibly give further insight into both the etiology and the prevention of this malignancy . Acidic pH induces topoisomerase II-mediated DNA damage . Acidic pH plays an important role in various pathophysiological states and has been demonstrated to be carcinogenic in animal models . Recent studies have also implicated acidic pH in the development of preneoplastic Barrett 's esophagus in human . However , little is known about the molecular mechanism underlying acidic pH-induced carcinogenesis . In the current study , we show that acidic pH , like the topoisomerase II ( P11388 ) poison DB00773 ( demethylepipodophyllotoxin ethylidene-beta-D-glucoside ) , induces tumors in 9,10-dimethyl-1,2-benzanthracene(DMBA)-initiated mice . The following studies in tissue culture models have suggested that acidic pH acts like a P11388 poison to induce P11388 -mediated DNA damage : ( i ) acidic pH induces P11388 -dependent DNA damage signals as evidenced by up-regulation of p53 and DB00133 -139 phosphorylation of P16104 [ a substrate for ataxia telangiectasia mutated ( Q13315 ) Q13315 and Rad3-related ( ATR ) kinases ] ; ( ii ) acidic pH-induced cytotoxicity in tumor cells is reduced in P11388 -deficient cells ; ( iii ) acidic pH increases the mutation frequency of the hypoxanthine phosphoribosyl transferase ( P00492 ) gene in a P11388 -dependent manner ; and ( iv ) acidic pH induces reversible P11388 -mediated DNA strand breaks in vitro . We discuss the possibility that P11388 -mediated DNA damage may contribute to acidic pH-induced carcinogenesis . Senescence-associated secretory phenotype in a mouse model of bleomycin-induced lung injury . DB00290 produces DNA damage , apoptosis and senescence , all of which play crucial roles in the development of pulmonary fibrosis . Recently , close attention has been paid to a DNA damage-induced phenotypic change ( senescence-associated secretory phenotype ; SASP ) as a trigger for the secretion of various mediators which modify the processes of tissue injury , inflammation , repair and fibrosis . We characterized the SASP in a murine model of bleomycin-induced lung injury . Mice were intratracheally administered bleomycin or control saline , and the lungs were obtained on days 7 , 14 and 21 . The occurrence of DNA damage and the SASP in the lungs was examined by immunostaining . γ P16104 immunostaining of the bleomycin-treated lungs revealed double-strand breaks ( DSBs ) , largely within P12830 -positive , β4-integirn-positive alveolar epithelial cells . The DSBs were associated with phosphorylation of Q13315 /ATR , a central signal transducer mediating the DNA damage response , and upregulation of the cyclin-dependent kinase inhibitor P38936 (CIP1) . The DSBs persisted for at least 21 days after the bleomycin exposure , although it began to wane after 7 days . A subpopulation of the γ P16104 -positive , DNA-damaged cells exhibited the SASP , characterized by overexpression of P05231 , TNFα , P08253 and P14780 , in association with the phosphorylation of IKKα/β and p38 MAPK . Persistent DNA damage and the SASP are induced in the process of bleomycin-induced lung injury and repair , suggesting that these events play an important role in the regulation of inflammation and tissue remodeling in bleomycin-induced pneumopathy . DB00338 , a gastric proton pump inhibitor , inhibits melanogenesis by blocking Q04656 trafficking . DB00338 is a proton pump inhibitor used in the treatment of peptic ulcer disease and gastrosophageal reflux disease and acts by irreversibly blocking P20648 , a P-type H+/K+ ATPase in gastric parietal cells . We found that omeprazole and its closely related congeners inhibited melanogenesis at micromolar concentrations in B16 mouse melanoma cells , normal human epidermal melanocytes , and in a reconstructed human skin model . DB00338 topically applied to the skin of UV-irradiated human subjects significantly reduced pigment levels after 3 weeks compared with untreated controls . DB00338 had no significant inhibitory effect on the activities of purified human tyrosinase or on the mRNA levels of tyrosinase , dopachrome tautomerase , Pmel17 , or O75030 mRNA levels . Although melanocytes do not express P20648 , they do express Q04656 , a copper transporting P-type ATPase in the trans-Golgi network that is required for copper acquisition by tyrosinase . Q04656 relocalization from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations in melanocytes was inhibited by omeprazole . DB00338 treatment increased the proportion of EndoH sensitive tyrosinase , indicating that tyrosinase maturation was impaired . In addition , omeprazole reduced tyrosinase protein abundance in the presence of cycloheximide , suggestive of increased degradation . Our findings are consistent with the hypothesis that omeprazole reduces melanogenesis by inhibiting Q04656 and by enhancing degradation of tyrosinase . DB00820 , a further innovation in the treatment of sexual dysfunction . In recognition of the large number of sufferers of sexual dysfunction worldwide , and the variety of etiologies of the condition , investigation into effective pharmacological agents has been expanded . One method of intervention is inhibition of the phosphodiesterase type 5 ( O76074 ) enzyme , which has already been exploited with a considerable degree -- though not complete -- success . A number of new agents that inhibit O76074 are under development . Notable among these is tadalafil , which has demonstrated a high level of selectivity for O76074 over the other phosphodiesterases and has shown efficacy in improving erectile function and sexual satisfaction in phase III trials . Throughout the clinical development program for tadalafil , the drug has been well tolerated and without serious side effects . The manufacturer , Lilly Q9Y6W8 , received an approvable letter from the US Food and Drug Administration for use of the drug as a treatment for erectile dysfunction on April 30 , 2002 . Lilly Q9Y6W8 hopes to market tadalafil , with the trade name DB00820 , in the USA in 2003 . A p53 axis regulates B cell receptor-triggered , innate immune system-driven B cell clonal expansion . Resting mature human B cells undergo a dynamic process of clonal expansion , followed by clonal contraction , during an in vitro response to surrogate C3d-coated Ag and innate immune system cytokines , P05112 and Q9Y275 . In this study , we explore the mechanism for clonal contraction through following the time- and division-influenced expression of several pro- and anti-apoptotic proteins within CFSE-labeled cultures . Several findings , involving both human and mouse B cells , show that a mitochondria-dependent apoptotic pathway involving p53 contributes to the high activation-induced cell death ( AICD ) susceptibility of replicating blasts . Activated B cell clones exhibit elevated p53 protein and elevated mRNA/protein of proapoptotic molecules known to be under direct p53 transcriptional control , Bax , Bad , Puma , Bid , and procaspase 6 , accompanied by reduced anti-apoptotic Bcl-2 . Under these conditions , Bim levels were not increased . The finding that full-length Bid protein significantly declines in AICD-susceptible replicating blasts , whereas Bid mRNA does not , suggests that Bid is actively cleaved to short-lived , proapoptotic truncated Bid . AICD was diminished , albeit not eliminated , by p53 small interfering RNA transfection , genetic deletion of p53 , or Bcl-2 overexpression . DNA damage is a likely trigger for p53-dependent AICD because susceptible lymphoblasts expressed significantly elevated levels of both phosphorylated ataxia telangiectasia mutated- DB00133 (1980) and phospho- P16104 - DB00133 (139) . Deficiency in activation-induced cytosine deaminase diminishes but does not ablate murine B cell AICD , indicating that activation-induced cytosine deaminase-induced DNA damage is only in part responsible . Evidence for p53-influenced AICD during this route of T cell-independent clonal expansion raises the possibility that progeny bearing p53 mutations might undergo positive selection in peripherally inflamed tissues with elevated levels of P05112 and Q9Y275 . Stimulation of epithelial repair is a likely mechanism for the action of mifepristone in reducing duration of bleeding in users of progestogen-only contraceptives . Many women using progestogen ( P ) -only contraceptives experience uterine bleeding problems . In clinical trials , a single low dose of mifepristone , given to DB00294 users at the beginning of a bleeding episode reduced the number of bleeding days by approximately 50 % compared with controls . In this study , a single dose of mifepristone was administered to etonogestrel ( P17813 ) -exposed pseudo-pregnant mice , 5 days after artificial decidualization was induced when the endometrium showed signs of bleeding . Control mice received vehicle alone . Mice were culled 12- , 18- , 24- and 48-h post-treatment . In the continued presence of P17813 , a single dose of mifepristone stimulated tissue breakdown followed by very rapid repair : most treated tissues were fully restored to the pre-decidualized state by 48 h post-treatment . During repair , proliferating cells ( Ki67 immunostained ) were localized to a band of cells around the basal area in breaking down tissues and to the repairing luminal epithelium and glands . P06401 -positive cells were largely localized to the basal area of the breaking down tissue in treated mice compared with decidual cells in controls . Oestrogen receptor-positive cells were observed in the repairing luminal epithelium and glands compared with the decidua and the basal region in control tissues . It is concluded that mifepristone treatment stimulates rapid restoration of luminal epithelial integrity : such action may be a key event in reducing the number of bleeding days observed in women using DB00294 who were treated with a single dose of mifepristone . A new role for the P40763 inhibitor , Q9Y6X2 : a repressor of microphthalmia transcription factor . In vitro and in vivo evidence suggest that microphthalmia transcription factor ( O75030 ) plays a key regulatory role in tissue-specific gene regulation in several cell types , including melanocytes , osteoclasts , and mast cells . A yeast two-hybrid search , using a portion of a nonmutated O75030 gene as the bait in the screening of a mast cell library , resulted in the isolation of the P40763 inhibitor , Q9Y6X2 . Q9Y6X2 is a transcriptional inhibitor that acts by specifically inhibiting P40763 's DNA binding activity . We found that it can directly associate with O75030 using an in vitro pull-down assay . Immunoprecipitation of O75030 from rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of Q9Y6X2 . Co-transfection of O75030 with Q9Y6X2 in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated up to 94 % inhibition of O75030 -mediated transcriptional activation . Using a gel-shift assay , it was shown that Q9Y6X2 can block DNA binding activity . It was also found that P40763 does not interfere , either in vitro or in vivo , with the interaction between Q9Y6X2 and O75030 . These data suggest that Q9Y6X2 functions in vivo as a key molecule in supressing the transcriptional activity of O75030 , a role of considerable importance in mast cell and melanocyte development .
[ "DB00072" ]
MH_train_1
MH_train_1
MH_train_1
interacts_with DB09079?
multiple_choice
[ "DB00294", "DB00313", "DB00588", "DB00755", "DB00783", "DB02546", "DB02901", "DB06822", "DB08910" ]
Prolonged treatment with bicalutamide induces androgen receptor overexpression and androgen hypersensitivity . BACKGROUND : Various hormone refractory prostate cancer cell models have been established with androgen depletion and have helped to clarify the mechanism for the transition into androgen-depletion independent status . However , the mechanism of bicalutamide resistance remains unclear because few cell models have been generated . METHODS : We generated a bicalutamide-resistant subline , LNCaP- O43633 , from LNCaP after prolonged treatment with bicalutamide . Androgen and/or bicalutamide responsiveness for proliferation and prostate-specific antigen ( PSA ) secretion were examined in vitro and in vivo . DB00624 and dihydrotestosterone ( DB02901 ) levels in xenografted tumors were analyzed by liquid chromatography-tandem mass spectrometry . P10275 ( AR ) gene mutation and amplification and AR and pAR(210) expression were determined . RESULTS : LNCaP- O43633 did not grow in an androgen-depleted medium and proliferation was stimulated in a tenfold lower concentration of androgen than that of LNCaP . LNCaP- O43633 grew in castrated male mice , and the DB02901 level in grafted LNCaP- O43633 tumors was 7.7-fold lower than in LNCaP tumors . DB01128 stimulated LNCaP- O43633 proliferation and PSA secretion in vitro and the antitumor activity of bicalutamide against LNCaP- O43633 was weaker than that of LNCaP in vivo . Additional AR mutation and AR gene amplification were not detected in LNCaP- O43633 , but AR and pAR(210) expression and PSA secretion in LNCaP- O43633 were higher than in LNCaP . CONCLUSIONS : DB01128 -resistant LNCaP- O43633 exhibited AR overexpression and hypersensitivity to low levels of androgen . Our data suggests that AR overexpression is a significant mechanism of bicalutamide resistance similar to resistance from chronic androgen depletion . In addition , pAR(210) overexpression could be a potential mechanism for hypersensitivity to low androgen in LNCaP- O43633 . Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries . BACKGROUND : Atherosclerosis affects aorta , coronary , carotid , and iliac arteries most frequently than any other body vessel . There may be common molecular pathways sustaining this process . Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis . RESULTS : We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression . We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis . Caveolae and JAK/ P35610 pathways , and P06702 / P05109 interacting proteins are certainly involved in the development of vascular disease . We found that the system of caveolae is directly connected with genes that respond to hormone receptors , and indirectly with the apoptosis pathway . Cytokines , chemokines and growth factors released in the blood flux were investigated in parallel . High levels of RANTES , IL-1ra , MIP-1 alpha , MIP-1 beta , P60568 , P05112 , P05113 , P05231 , P13232 , Q16552 , DB00102 , P15692 and P01579 were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels . CONCLUSION : The pattern of cytokine and P06702 / P05109 up-regulation characterizes atherosclerosis as a proinflammatory disorder . Activation of the JAK/ P35610 pathway is confirmed by the up-regulation of P05231 , P42224 , Q00978 and Q13651 genes in coronary and carotid plaques . The functional network constructed in our research is an evidence of the central role of P35610 protein and the caveolae system to contribute to preserve the plaque . Moreover , Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype . Targeting Q01196 / Q06455 -histone deacetylase repressor complex : a novel mechanism for valproic acid-mediated gene expression and cellular differentiation in Q01196 / Q06455 -positive acute myeloid leukemia cells . In t(8;21) acute myeloid leukemia ( AML ) , the Q01196 / Q06455 fusion protein promotes leukemogenesis by recruiting class I histone deacetylase ( HDAC ) -containing repressor complex to the promoter of Q01196 target genes . Valproic acid ( DB00313 ) , a commonly used antiseizure and mood stabilizer drug , has been shown to cause growth arrest and induce differentiation of malignant cells via HDAC inhibition . DB00313 causes selective proteasomal degradation of Q92769 but not other class I HDACs ( i.e. , HDAC 1 , 3 , and 8 ) . Therefore , we raised the question of whether this drug can effectively target the leukemogenic activity of the Q01196 / Q06455 fusion protein that also recruits Q13547 , a key regulator of normal and aberrant histone acetylation . We report here that DB00313 treatment disrupts the Q01196 / Q06455 - Q13547 physical interaction , stimulates the global dissociation of Q01196 / Q06455 - Q13547 complex from the promoter of Q01196 / Q06455 target genes , and induces relocation of both Q01196 / Q06455 and Q13547 protein from nuclear to perinuclear region . Furthermore , we show that mechanistically these effects associate with a significant inhibition of HDAC activity , histone H3 and H4 hyperacetylation , and recruitment of RNA polymerase II , leading to transcriptional reactivation of target genes ( i.e. , P08700 ) otherwise silenced by Q01196 / Q06455 fusion protein . Ultimately , these pharmacological effects resulted in significant antileukemic activity mediated by partial cell differentiation and caspase-dependent apoptosis . Taken together , these data support the notion that DB00313 might effectively target Q01196 / Q06455 -driven leukemogenesis through disruption of aberrant Q13547 function and that DB00313 should be integrated in novel therapeutic approaches for Q01196 / Q06455 -positive AML . Glucocorticoid-induced surface expression of annexin 1 blocks beta2-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 ) . 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 . METHODS : Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate ( FP ) , and beta2-integrin adhesion was measured after stimulation with P05113 or eotaxin . Effects of FP on P47712 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 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 beta2-integrin adhesion . Translocation of P47712 to the nuclear membrane was significantly blocked by incubation with FP . Blockade was reversed with annexin 1 blocking antibody . CONCLUSION : Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1 , which blocks P47712 translocation to nuclear membrane . P10275 YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration . X-linked spinal and bulbar muscular atrophy ( SBMA ) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration . SBMA is caused by polyglutamine repeat expansions in the androgen receptor ( AR ) . To determine the basis of AR polyglutamine neurotoxicity , we introduced human AR yeast artificial chromosomes carrying either 20 or 100 CAGs into mouse embryonic stem cells . The AR100 transgenic mice developed a late-onset , gradually progressive neuromuscular phenotype accompanied by motor neuron degeneration , indicating striking recapitulation of the human disease . We then tested the hypothesis that polyglutamine-expanded AR interferes with CREB binding protein ( CBP ) -mediated transcription of vascular endothelial growth factor ( P15692 ) and observed altered CBP-AR binding and P15692 reduction in AR100 mice . We found that mutant AR-induced death of motor neuron-like cells could be rescued by P15692 . Our results suggest that SBMA motor neuronopathy involves altered expression of P15692 , consistent with a role for P15692 as a neurotrophic/survival factor in motor neuron disease . Expression of vitamin D3 receptor and retinoid receptors in human breast cancer : identification of potential heterodimeric receptors . DB00169 ( VD ) and all-trans-retinoic acid ( DB00755 ) have been postulated as a novel treatment option for breast carcinoma . Since the combined effects of retinoids and VD derivatives are attributed to heterodimeric interactions between members of the nuclear receptor family , the expression patterns of the heterodimers formed by vitamin D3 receptor ( P11473 ) and the retinoid receptors RARs ( P10276 , P10826 and P13631 ) and RXRs ( RXR-alpha , RXR-beta and RXR-gamma ) have been studied by immunohistochemistry in benign and malignant breast tissues . Present results revealed that immunoexpressions to all receptor types studied were higher in both in situ and infiltrative carcinomas than in benign breast diseases . In a variable number of cases of infiltrative carcinoma , immunostaining appeared in the nucleus , whereas in the other two disorders immunostaining was only cytoplasmic . The correlation established between P11473 and the different isoforms of retinoid receptors revealed that P11473 seems to select mainly P10276 to form heterodimers and to exert their properties as transcription factor . The results of this study suggest that this heterodimer plays a critical role in cancer malignancy , and its presence indicates those patient groups presenting a better response to adjuvant therapies based on the combination of vitamin D and DB00755 . DB00184 induces cell proliferation , invasion and epithelial-mesenchymal transition in a variety of human cancer cell lines . Cigarette smoking is strongly correlated with the onset of nonsmall cell lung cancer ( NSCLC ) . DB00184 , an active component of cigarettes , has been found to induce proliferation of lung cancer cell lines . In addition , nicotine can induce angiogenesis and confer resistance to apoptosis . All these events are mediated through the nicotinic acetylcholine receptors ( nAChRs ) on lung cancer cells . In this study , we demonstrate that nicotine can promote anchorage-independent growth in NSCLCs . In addition , nicotine also induces morphological changes characteristic of a migratory , invasive phenotype in NSCLCs on collagen gel . These morphological changes were similar to those induced by the promigratory growth factor P15692 . The proinvasive effects of nicotine were mediated by alpha7-nAChRs on NSCLCs . RT-PCR analysis showed that the alpha7-nAChRs were also expressed on human breast cancer and pancreatic cancer cell lines . DB00184 was found to promote proliferation and invasion in human breast cancer . The proinvasive effects of nicotine were mediated via a nAChR , Src and calcium-dependent signaling pathway in breast cancer cells . In a similar fashion , nicotine could also induce proliferation and invasion of Aspc1 pancreatic cancer cells . Most importantly , nicotine could induce changes in gene expression consistent with epithelial to mesenchymal transition ( EMT ) , characterized by reduction of epithelial markers like P12830 expression , ZO-1 staining and concomitant increase in levels of mesenchymal proteins like vimentin and fibronectin in human breast and lung cancer cells . Therefore , it is probable that the ability of nicotine to induce invasion and EMT may contribute to the progression of breast and lung cancers . Identification of an acetylation-dependant P12956 /FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis . FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors . We report a novel interaction between FLIP and the DNA repair protein P12956 that regulates FLIP protein stability by inhibiting its polyubiquitination . Furthermore , we found that the histone deacetylase ( HDAC ) inhibitor DB02546 ( DB02546 ) enhances the acetylation of P12956 , thereby disrupting the FLIP/ P12956 complex and triggering FLIP polyubiquitination and degradation by the proteasome . Using in vitro and in vivo colorectal cancer models , we further demonstrated that DB02546 -induced apoptosis is dependant on FLIP downregulation and caspase 8 activation . In addition , an Q9UBN7 -specific inhibitor Tubacin recapitulated the effects of DB02546 , suggesting that Q9UBN7 is a key regulator of P12956 acetylation and FLIP protein stability . Thus , HDAC inhibitors with anti- Q9UBN7 activity act as efficient post-transcriptional suppressors of FLIP expression and may , therefore , effectively act as ' FLIP inhibitors ' . DB00107 alleviates the neuroendocrine and cytokine response to bacterial endotoxin in healthy men . DB00107 is a hormone and neurotransmitter found to have anti-inflammatory functions in rodents . Here we used experimental bacterial endotoxinemia to examine the role of exogenous oxytocin administration on innate immune responses in humans . Ten healthy men received , in a randomized , placebo-controlled , crossover design , placebo , oxytocin , LPS , and LPS + oxytocin . DB00107 treatment resulted in a transient or prolonged reduction of endotoxin-induced increases in plasma DB01285 , cortisol , procalcitonin , P01375 , IL-1 receptor antagonist , P05112 , P05231 , macrophage inflammatory protein-1alpha , macrophage inflammatory protein-1beta , monocyte chemoattractant protein-1 ( P13500 ) , interferon-inducible protein 10 , and P15692 . In vitro , oxytocin had no impact on LPS effects in releasing P01375 , P05231 , and P13500 in monocytes and peripheral blood mononuclear cells from healthy human donors . In summary , oxytocin decreases the neuroendocrine and cytokine activation caused by bacterial endotoxin in men , possibly due to the pharmacological modulation of the cholinergic anti-inflammatory pathway . DB00107 might be a candidate for the therapy of inflammatory diseases and conditions associated with high cytokine and P15692 levels . Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo . Expression of the chemokine receptor P61073 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express P48061 . In this study , we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides . Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I(125) P48061 . DB01109 dodecasaccharides were found to be the minimal chain length required to efficiently bind P48061 ( 71 % inhibition ; P < 0.001 ) . These oligosaccharides also significantly inhibited P48061 -induced migration of P61073 -expressing LMD MDA-MB 231 breast cancer cells . In addition , heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product , DB06822 . When given subcutaneously in a SCID mouse model of human breast cancer , heparin dodecasaccharides had no effect on the number of lung metastases , but did however inhibit ( P < 0.05 ) tumour growth ( lesion area ) compared to control groups . In contrast , polymeric heparin significantly inhibited both the number ( P < 0.001 ) and area of metastases , suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases . Modeling the neurovascular niche : murine strain differences mimic the range of responses to chronic hypoxia in the premature newborn . Preterm birth results in significant cognitive and motor disabilities , but recent evidence suggests that there is variable recovery over time . One possibility that may explain this variable recovery entails variable neurogenic responses in the subventricular zone ( SVZ ) following the period of chronic hypoxia experienced by these neonates . In this report , we have characterized the responses to chronic hypoxia of two mouse strains that represent a wide range of susceptibility to chronic hypoxia . We determined that C57BL/6 pups and neural progenitor cells ( NPCs ) derived from them exhibit a blunted response to hypoxic insult compared with CD-1 pups and NPCs . Specifically , C57BL/6 pups and NPCs exhibited blunted in vivo and in vitro proliferative and increased apoptotic responses to hypoxic insult . Additionally , C57BL/6 NPCs exhibited lower baseline levels and hypoxia-induced levels of selected transcription factors , growth factors , and receptors ( including HIF-1alpha , Q9GZT9 , P23560 , P15692 , P48061 , TrkB , Nrp-1 , P61073 , and NO ) that determine , in part , the responsiveness to chronic hypoxic insult compared with CD-1 pups and NPCs , providing insight into this important and timely problem in perinatology . 4-[3,5-Bis(trimethylsilyl)benzamido] benzoic acid inhibits angiogenesis in colon cancer through reduced expression of vascular endothelial growth factor . 4-[3,5-bis(trimethylsilyl)benzamido] Benzoic acid ( TAC-101 ) has potent antiproliferative , antiangiogenic , and antitumor effects in vitro and in vivo . These effects might be due to TAC-101 binding to retinoic acid receptor alpha ( P10276 ) and interfering with the binding of activator protein-1 ( AP-1 ) to DNA . However , little is known about the detailed mechanism of TAC-101 function . We investigated the mechanism of the antiangiogenic effect of TAC-101 using a rat hepatic metastatic model in vivo and DLD-1 human colon cancer cells in vitro . Liver metastases were induced by portal injection of Q15293 -9 rat colonic cancer cells into F344 rats . TAC-101 ( 8 mg/kg ) was orally administered 5 days per week for 4 weeks and then hepatic tumors were immunohistochemically evaluated for microvessel density ( P53602 ) and vascular endothelial growth factor ( P15692 ) . TAC-101 significantly reduced both P53602 and P15692 expression . Northern blot analysis and ELISA indicated that TAC-101 efficiently inhibited production of P15692 mRNA and protein in DLD-1 cells in a time- and dose-dependent manner . These findings suggest that TAC-101 may inhibit progression and metastasis in colon cancer by interfering with tumor production of P15692 . A curated database of miRNA mediated feed-forward loops involving MYC as master regulator . BACKGROUND : The MYC transcription factors are known to be involved in the biology of many human cancer types . But little is known about the Myc/microRNAs cooperation in the regulation of genes at the transcriptional and post-transcriptional level . METHODOLOGY/PRINCIPAL FINDINGS : Employing independent databases with experimentally validated data , we identified several mixed microRNA/Transcription Factor Feed-Forward Loops regulated by Myc and characterized completely by experimentally supported regulatory interactions , in human . We then studied the statistical and functional properties of these circuits and discussed in more detail a few interesting examples involving Q01094 , P60484 , P06400 and P15692 . CONCLUSIONS/SIGNIFICANCE : We have assembled and characterized a catalogue of human mixed Transcription Factor/microRNA Feed-Forward Loops , having Myc as master regulator and completely defined by experimentally verified regulatory interactions . DB09079 , a triple angiokinase inhibitor , enhances cytotoxic therapy response in pancreatic cancer . Angiogenesis remains a sensible target for pancreatic ductal adenocarcinoma ( PDAC ) therapy . P15692 , PDGF , FGF and their receptors are expressed at high levels and correlate with poor prognosis in human PDAC . DB09079 is a triple angiokinase inhibitor that targets P17948 /2/3 , P11362 /2/3 and PDGFRα/β signaling . We investigated the antitumor activity of nintedanib alone or in combination with the cytotoxic agent gemcitabine in experimental PDAC . DB09079 inhibited proliferation of cells from multiple lineages found in PDAC , with gemcitabine enhancing inhibitory effects . DB09079 blocked PI3K/MAPK activity and induced apoptosis in vitro and in vivo . In a heterotopic model , net local tumor growth compared to controls ( 100 % ) was 60.8 ± 10.5 % in the gemcitabine group , -2.1 ± 9.9 % after nintedanib therapy and -12.4 ± 16 % after gemcitabine plus nintedanib therapy . Effects of therapy on intratumoral proliferation , microvessel density and apoptosis corresponded with tumor growth inhibition data . In a PDAC survival model , median animal survival after gemcitabine , nintedanib and gemcitabine plus nintedanib was 25 , 31 and 38 days , respectively , compared to 16 days in controls . The strong antitumor activity of nintedanib in experimental PDAC supports the potential of nintedanib-controlled mechanisms as targets for improved clinical PDAC therapy . Q00987 is a ubiquitin ligase of P12956 -Akt promotes cell survival by inhibiting Q00987 -dependent P12956 destabilization . Earlier , we have reported that 70 kDa subunit of Ku protein heterodimer ( P12956 ) binds and inhibits Bax activity in the cytosol and that ubiquitin ( Ub ) -dependent proteolysis of cytosolic P12956 facilitates Bax-mediated apoptosis . We found that Q00987 ( human homolog of murine double minute ) has an ability to ubiquitinate P12956 and that Q00987 overexpression in cultured cells causes a decrease in P12956 expression levels . An interaction between P12956 and Q00987 was shown by means of immunoprecipitation , whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Q00987 . Vascular endothelial growth factor ( P15692 ) is known to inhibit endothelial cell ( EC ) apoptosis through an Akt-mediated survival kinase signal ; however , the mechanism underlying this inhibition of apoptosis has not been fully elucidated . We found that P15692 inhibited cytosolic P12956 degradation induced by apoptotic stress . It is known that Akt-dependent phosphorylation of Q00987 causes nuclear translocation of Q00987 followed by Q00987 -mediated inactivation of p53 . We found that P15692 stimulated nuclear translocation of Q00987 in EC and efficiently inhibited P12956 degradation . We also found that constitutively active Akt , but not kinase-dead Akt , inhibited P12956 degradation in the cytosol . Furthermore , P12956 knockdown diminished antiapoptotic activity of Akt . Taken together , we propose that Q00987 is a P12956 Ub ligase and that Akt inhibits Bax-mediated apoptosis , at least in part , by maintaining P12956 levels through the promotion of Q00987 nuclear translocation . Pharmacological properties of thalidomide and its analogues . Thalidomide and its immunomodulatory imide drugs ( IMiDs ) analogues DB00480 ( DB00480 , DB00480 ) and CC-4047 ( Actimid , DB08910 ) have been used as anti-inflammatory and anticancerous drugs in the recent years . Thalidomide and IMiDs inhibit the cytokines tumour necrosis factor-alpha ( P01375 ) , interleukins ( IL ) 1-beta , 6 , 12 , and granulocyte macrophage-colony stimulating factor ( GM- P04141 ) . They also costimulate primary human T , NKT and NK lymphocytes inducing their proliferation , cytokine production , and cytotoxic activity . On the other hand , the compounds are anti-angiogenic , anti-proliferative , and pro-apoptotic . Thalidomide analogues have been used as inhibitors of alpha glucosidase and could be potential drugs for diabetes treatment . In this review , we explore the current trend of the different structures , the new patents , and the possible new applications in different pathologies . Loss of epigenetic Kruppel-like factor 4 histone deacetylase ( KLF-4-HDAC ) -mediated transcriptional suppression is crucial in increasing vascular endothelial growth factor ( P15692 ) expression in breast cancer . Vascular endothelial growth factor ( P15692 ) is recognized as an important angiogenic factor that promotes angiogenesis in a series of pathological conditions , including cancer , inflammation , and ischemic disorders . We have recently shown that the inflammatory transcription factor P56270 is , at least in part , responsible for the marked increase of P15692 levels in breast cancer . Here , we show that P56270 -mediated induction of P15692 is repressed by KLF-4 transcription factor . KLF-4 is abundantly present in normal breast epithelial cells , but its level is considerably reduced in breast cancer cells and clinical cancer tissues . In the human P15692 promoter , P56270 - and KLF-4-binding elements are overlapping , whereas P56270 induces and KLF-4 suppresses P15692 expression . Ectopic overexpression of KLF-4 and RNAi-mediated inhibition of endogenous KLF-4 supported the role of KLF-4 as a transcriptional repressor of P15692 and an inhibitor of angiogenesis in breast cancer cells . We show that KLF-4 recruits histone deacetylases ( HDACs ) -2 and -3 at the P15692 promoter . Chronological ChIP assays demonstrated the occupancy of KLF-4 , Q92769 , and O15379 in the P15692 promoter in normal MCF-10A cells but not in MDA-MB-231 cancer cells . Co-transfection of KLF-4 and HDAC expression plasmids in breast cancer cells results in synergistic repression of P15692 expression and inhibition of angiogenic potential of these carcinoma cells . Together these results identify a new mechanism of P15692 up-regulation in cancer that involves concomitant loss of KLF-4-HDAC-mediated transcriptional repression and active recruitment of P56270 -mediated transcriptional activation . Gender difference in the activity but not expression of estrogen receptors alpha and beta in human lung adenocarcinoma cells . The higher frequency of lung adenocarcinoma in women smokers than in men smokers suggests a role for gender-dependent factors in the etiology of lung cancer . We evaluated estrogen receptor ( ER ) alpha and beta expression and activity in human lung adenocarcinoma cell lines and normal lung fibroblasts . Q8N1N2 -length ERalpha and ERbeta proteins were expressed in all cell lines with higher ERbeta than ERalpha . Although estradiol ( E(2) ) binding was similar , E(2) stimulated proliferation only in cells from females , and this response was inhibited by anti-estrogens 4-hydroxytamoxifen ( DB04468 ) and DB00947 . In contrast , E(2) did not stimulate replication of lung adenocarcinoma cells from males and DB04468 or ICI did not block cell proliferation . Similarly , transcription of an estrogen response element-driven reporter gene was stimulated by E(2) in lung adenocarcinoma cells from females , but not males . P06401 ( PR ) expression was increased by E(2) in two out of five adenocarcinoma cell lines from females , but none from males . E(2) decreased P12830 protein expression in some of the cell lines from females , as it did in MCF-7 breast cancer cells , but not in the cell lines from males . Thus , ERalpha and ERbeta expression does not correlate with the effect of ER ligands on cellular activities in lung adenocarcinoma cells . On the other hand , coactivator Q15648 expression was higher in lung adenocarcinoma cells from females versus males and higher in adenocarcinoma cells than in normal human bronchial epithelial cells . Q15648 and other ER coregulators may contribute to differences in estrogen responsiveness between lung adenocarcinoma cells in females and males . Stimulation of epithelial repair is a likely mechanism for the action of mifepristone in reducing duration of bleeding in users of progestogen-only contraceptives . Many women using progestogen ( P ) -only contraceptives experience uterine bleeding problems . In clinical trials , a single low dose of mifepristone , given to DB00294 users at the beginning of a bleeding episode reduced the number of bleeding days by approximately 50 % compared with controls . In this study , a single dose of mifepristone was administered to etonogestrel ( P17813 ) -exposed pseudo-pregnant mice , 5 days after artificial decidualization was induced when the endometrium showed signs of bleeding . Control mice received vehicle alone . Mice were culled 12- , 18- , 24- and 48-h post-treatment . In the continued presence of P17813 , a single dose of mifepristone stimulated tissue breakdown followed by very rapid repair : most treated tissues were fully restored to the pre-decidualized state by 48 h post-treatment . During repair , proliferating cells ( Ki67 immunostained ) were localized to a band of cells around the basal area in breaking down tissues and to the repairing luminal epithelium and glands . P06401 -positive cells were largely localized to the basal area of the breaking down tissue in treated mice compared with decidual cells in controls . Oestrogen receptor-positive cells were observed in the repairing luminal epithelium and glands compared with the decidua and the basal region in control tissues . It is concluded that mifepristone treatment stimulates rapid restoration of luminal epithelial integrity : such action may be a key event in reducing the number of bleeding days observed in women using DB00294 who were treated with a single dose of mifepristone . 17 DB00783 -mediated growth inhibition of MDA-MB-468 cells stably transfected with the estrogen receptor : cell cycle effects . P03372 ( ER ) -negative MDA-MB-468 human breast cancer cells were stably transfected with wild-type human ER and utilized as a model for investigating estrogen- and aryl hydrocarbon ( Ah ) -responsiveness . Treatment of the stably transfected cells with 10 nM 17 beta-estradiol ( E2 ) resulted in a significant inhibition ( > 60 % ) of cell proliferation and DNA synthesis , which was blocked by 10(-7) M ICI 182 780 . Analysis by flow cytometry indicated that treatment with E2 increased the percentage of cells in G0/ P55008 ( from 68.8 to 89.4 ) and decreased cells in S ( from 18.4 to 3.4 ) and G2/M ( from 12.8 to 7.2 ) phases of the cell cycle . The effects of E2 on the major cyclins , cyclin-dependent kinases and cyclin-dependent kinase inhibitors , retinoblastoma protein ( RB ) , Q01094 , and cyclin-dependent kinase activities were also investigated in the stably transfected MDA-MB-468 cells . The results demonstrated that the growth inhibitory effects of 10(-8) M E2 in ER stably transfected MDA-MB-468 cells were associated with modulation of several factors required for cell cycle progression and DNA synthesis , including significant induction of the cyclin-dependent kinase inhibitor p21cip-1 ( > 4-fold increase after 12 h ) and decreased Q01094 and P12004 protein levels . These results show that the growth-inhibitory effects of E2 in the stably transfected cells were due to multiple factors which result in growth arrest in G0/ P55008 and inhibition of DNA synthesis .
[ "DB06822" ]
MH_train_2
MH_train_2
MH_train_2
interacts_with DB00083?
multiple_choice
[ "DB00035", "DB00293", "DB00322", "DB00341", "DB00351", "DB00588", "DB00741", "DB00951", "DB01200" ]
Anti-Parkinson 's disease drugs and pharmacogenetic considerations . INTRODUCTION : The development of pharmacogenetic-based clinical practice guidelines for the use of anti-Parkinson 's disease drugs requires , as a pre-requisite , the identification and validation of genetic biomarkers . These biomarkers are then used as surrogate endpoints . This review analyzes potential genetic biomarkers which can be used to improve anti-Parkinson 's disease therapy . AREAS COVERED : The authors present an overview of current knowledge of pharmacogenetic implications of anti-Parkinson 's disease drugs , including genes coding for the corresponding drug-metabolizing enzymes and drug targets . The gene/drug pairings with the strongest potential for pharmacogenetic recommendations include : P33261 /benztropine , P21964 /levodopa and entacapone , P20813 /selegiline , P22309 /entacapone , P14416 /ropinirole , pramipexole and cabergoline , and P35462 /ropinirole and pramipexole . Evidence supporting the effect of substrates , inhibitor or inducers for drug specific metabolizing enzymes in anti-Parkinson 's disease drug response includes P05177 in the response to ropinirole and rasagiline , and P08684 in the response to bromocriptine , lisuride , pergolide and cabergoline . The authors present and discuss the current information on gene variations according to the 1000 genomes catalog and other databases with regards to anti-Parkinson 's disease drugs . They also review and discuss the clinical implications of these variations . EXPERT OPINION : The goal of pharmacogenomic testing for anti-Parkinson 's disease drugs should be conservative and aimed at selecting determined drugs for determined patients . However , much additional research is still needed to obtain reliable pre-prescription tests . Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease . Botulinum neurotoxin ( BoNT ) metalloproteases and related proteases are the most selective proteases known . X-ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding . In order to characterize the postulated substrate-induced conformational changes for enzyme activation , we synthesized a series of transition-state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence . In this paper , we report our efforts to design inhibitors of DB00083 metalloprotease . We confirm that an effective substrate sequence for DB00083 metalloprotease is a 17-mer peptide corresponding to residues 187-203 of P60880 . A more stable substrate , Nle202SNAP-25 [ 187-203 ] was synthesized in order to develop an assay for proteolytic activity of DB00083 metalloprotease that can be used to monitor time-dependent inhibition . Alpha-thiol amide analogs of Gln-197 were incorporated via solid-phase peptide synthesis into both 17-mer minimal peptide substrate sequences . The synthesis , characterization and inhibition kinetics for the alpha-thiol amide analogs of holotoxin A substrate are described . These substrate-derived inhibitors were shown to be submicromolar inhibitors of DB00083 catalytic activity . Comparison of the catalytic properties of the botulinum neurotoxin subtypes A1 and A5 . Clostridium botulinum neurotoxins ( BoNTs ) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential SNARE proteins . There are seven serologically distinctive types of BoNTs and many subtypes within a serotype have been identified . BoNT/A5 is a recently discovered subtype of type A botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied A1 subtype . In the present study , we examined the endopeptidase activity of these two DB00083 subtypes and our results revealed significant differences in substrate binding and cleavage efficiency between subtype A5 and A1 . Distinctive hydrolysis efficiency was observed between the two toxins during cleavage of the native substrate P60880 versus a shortened peptide mimic . N-terminal truncation studies demonstrated that a key region of the P60880 , including the amino acid residues at 151 through 154 located in the remote binding region of the substrate , contributed to the differential catalytic properties between A1 and A5 . Elevated binding affinity of the peptide substrate resulted from including these important residues and enhanced BoNT/A5 's hydrolysis efficiency . In addition , mutations of these amino acid residues affect the proteolytic performance of the two toxins in different ways . This study provides a better understanding of the biological activity of these toxins , their performance characteristics in the Endopep-MS assay to detect BoNT in clinical samples and foods , and is useful for the development of peptide substrates . Comparative role of neurotoxin-associated proteins in the structural stability and endopeptidase activity of botulinum neurotoxin complex types A and E . Seven serotypes of botulinum neurotoxins , the most toxic substances known to mankind , are each produced by different strains of Clostridium botulinum along with a group of neurotoxin-associated proteins ( NAPs ) . NAPs play a critical role in the toxicoinfection process of botulism in addition to their role in protecting the neurotoxin from proteolytic digestion in the GI tract as well as from adverse environmental conditions . In this study we have investigated the effect of temperature on the structural and functional stability of DB00083 complex ( BoNT/AC ) and BoNT/E complex ( BoNT/EC ) . Although the NAPs in the two complexes are quite different , both groups of NAPs activate the endopeptidase activities of their BoNTs without any need to reduce the disulfide bonds between light and heavy chains of respective BoNTs . BoNT/AC attains optimum enzyme activity at the physiological temperature of 37 degrees C whereas BoNT/EC is maximally active at 45 degrees C , and this is accompanied by conformational alterations in its polypeptide folding at this temperature , leading to favorable binding with its intracellular substrate , P60880 , and subsequent cleavage of the latter . DB00083 in its complex form is found to be structurally more stable against temperature whereas BoNT/E in its complex form is functionally better protected against temperature . Based on the analysis of isolated NAPs we have observed that the structural stability of the BoNT/AC is contributed by the NAPs . In addition to the unique structural conditions in which the enzyme remains active , functional stability of botulinum neurotoxins against temperature plays a critical role in the survival of the agent in cooked food and in food-borne botulism . New insights into clostridial neurotoxin-SNARE interactions . Botulinum neurotoxin serotype A ( DB00083 ) has achieved a dichotomous status in modern medicine ; it is both a versatile treatment for several neurological disorders and a lethal poison responsible for causing the neuroparalytic syndrome botulism . The extent of paralysis largely depends on the dosage of toxin received . The toxins block neurotransmitter release by delivering their DB01593 (2+)-dependent protease components to the presynaptic side of chemical synapses . These highly specialized enzymes exclusively hydrolyze peptide bonds within SNARE ( soluble N-ethylmaleiamide-sensitive factor attachment protein receptor ) proteins . Recently , the structural basis for the highly specific interaction between DB00083 and its target SNARE , P60880 ( synaptosomal-associated protein of 25kDa ) , was elucidated . New details regarding the nature of the toxin-SNARE interactions could be exploited for novel inhibitor design . Second generation steroidal 4-aminoquinolines are potent , dual-target inhibitors of the botulinum neurotoxin serotype A metalloprotease and P. falciparum malaria . Significantly more potent second generation 4-amino-7-chloroquinoline ( 4,7-ACQ ) based inhibitors of the botulinum neurotoxin serotype A ( DB00083 ) light chain were synthesized . Introducing an amino group at the C(3) position of the cholate component markedly increased potency ( IC50 values for such derivatives ranged from 0.81 to 2.27 μM ) . Two additional subclasses were prepared : bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives ; both classes provided inhibitors with nanomolar-range potencies ( e.g. , the Ki of compound 67 is 0.10 μM ) . During DB00083 challenge using primary neurons , select derivatives protected P60880 by up to 89 % . Docking simulations were performed to rationalize the compounds ' in vitro potencies . In addition to specific residue contacts , coordination of the enzyme 's catalytic zinc and expulsion of the enzyme 's catalytic water were a consistent theme . With respect to antimalarial activity , the compounds provided better IC90 activities against chloroquine resistant ( CQR ) malaria than CQ , and seven compounds were more active than mefloquine against CQR strain W2 . P01375 -alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes . OBJECTIVE : It has been demonstrated that tumor necrosis factor-alpha ( P01375 alpha ) induces apoptosis in cardiac myocytes . However , its mechanism of action is still not well understood . In the present study , we hypothesized that P01375 alpha induces myocardial apoptosis by induction of inducible nitric oxide synthase ( P35228 ) . METHODS : Neonatal cardiac myocytes were isolated from P35228 ( -/- ) mutant and C57BL6 wild type mice . Cells were cultured for 3 days before treatment with an NO donor or P01375 alpha . Following treatment with S-nitroso-N-acetyl-penicillamine ( P60880 ) or P01375 , cells were tested for apoptosis by terminal deoxynucleotidyl transfer-mediated end labeling ( TUNEL ) staining and cell death detection ELISA . NO production was measured by nitrite concentration in the culture medium . Cardiomyocyte expression of P35228 and P01375 type 1 receptor ( P19438 ) mRNA was determined by reverse transcriptase-polymerase chain reaction ( RT-PCR ) . RESULTS : P60880 ( 0.01-100 microM ) induced apoptosis of cardiac myocytes in a concentration-dependent manner in the wild type mice ( n = 5 , P < 0.01 ) . P19438 mRNA was expressed in neonatal cardiomyocytes from both wild type and P35228 ( -/- ) mutant mice . P01375 alpha induced a concentration-dependent increase in P35228 mRNA expression and nitrite production as well as significant apoptosis of cardiomyocytes in the wild type mice ( n = 4 , P < 0.01 ) . However , without P35228 expression , the apoptotic effects of P01375 were significantly attenuated in cardiomyocytes from P35228 ( -/- ) mutant mice ( n = 4 , P < 0.05 ) . CONCLUSION : P01375 alpha induces apoptosis via P35228 expression and NO production in neonatal mouse cardiomyocytes . Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins . Rat brain synaptosomes were used to study the effect of several clostridial neurotoxins on the neurotransmitter release . In this system the blockade of transmitter release correlated with the proteolytic activity of the toxins . Blockade of glutamate release was linked to selective proteolysis of one of the following synaptic proteins : synaptobrevin ( BoNT/D , BoNT/F ) ; P60880 ( DB00083 , BoNT/E ) , or HPC-1/syntaxin ( BoNT/C1 ) . All the toxins used had an inhibitory effect on synaptosomes with the exception of BoNT/F . BoNT/F cleaved synaptobrevin in permeabilized synaptosomes but failed to produce the same effect on intact synaptosomes . Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease : implications for dual substrate specificity . Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus . They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors ( SNAREs ) P60880 , syntaxin , and synaptobrevin , which constitute part of the synaptic vesicle fusion machinery . The catalytic component of the clostridial neurotoxins is their light chain ( LC ) , a DB01593 endopeptidase . There are seven structurally and functionally related botulinum neurotoxins ( BoNTs ) , termed serotype A to G , and tetanus neurotoxin ( TeNT ) . Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave . The mechanisms of action for substrate recognition and target cleavage are largely unknown . Here , we report structural and biochemical studies of BoNT/C1-LC , which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and P60880 . A distinct pocket ( S1 ' ) near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1 ' residue for both P60880 and syntaxin . Mutations of this P60880 residue dramatically reduce enzymatic activity . The remote alpha-exosite that was previously identified in the complex of DB00083 -LC and P60880 is structurally conserved in BoNT/C1 . However , mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of DB00083 , which could account for its dual substrate specificity . Agonism at P41595 receptors is not a class effect of the ergolines . Restrictive cardiac valvulopathies observed in Parkinson patients treated with the ergoline dopamine agonist pergolide have recently been associated with the agonist efficacy of the drug at 5-hydroxytryptamine2B ( P41595 ) receptors . To evaluate whether agonism at P41595 receptors is a phenomenon of the class of the ergolines , we studied P41595 receptor-mediated relaxation in porcine pulmonary arteries to five ergolines which are used as antiparkinsonian drugs . DB01186 and cabergoline were potent full agonists in this tissue ( pEC50 8.42 and 8.72 ) . DB01200 acted as a partial agonist ( pEC50 6.86 ) . Lisuride and terguride , however , failed to relax the arteries but potently antagonized 5-HT-induced relaxation ( pKB 10.32 and 8.49 ) . Thus , agonism at P41595 receptors seems not to be a class effect of the ergolines . Botulinum neurotoxin A activity is dependent upon the presence of specific gangliosides in neuroblastoma cells expressing synaptotagmin I . Botulinum neurotoxin A ( DB00083 ) is the deadliest of all known biological substances . Although its toxicity makes DB00083 a biological warfare threat , its biologic activity makes it an increasingly useful therapeutic agent for the treatment of muscular disorders . However , almost 200 years after its discovery , the neuronal cell components required for the activity of this deadly toxin have not been unequivocally identified . In this work , neuroblastoma cells expressing synaptotagmin I , a protein shown to be bound by DB00083 , were used to determine whether specific gangliosides were necessary for DB00083 activity as measured by synaptosomal-associated protein of 25 kDa ( P60880 ) cleavage . Ganglioside GT1b was found to support DB00083 activity significantly more effectively than GD1a , which was far more effective than GM1 when added to ganglioside-deficient murine cholinergic Neuro 2a or to human adrenergic SK-N-SH neuroblastoma cells . Whereas both cell lines expressed synaptotagmin I , P60880 cleavage was not observed in the absence of complex gangliosides . These results indicate that 1 ) gangliosides are required for DB00083 activity , 2 ) synaptotagmin I in the absence of gangliosides does not support DB00083 activity , and 3 ) Neuro 2a cells are an efficient model system for studying the biological activity of DB00083 . Inhibition of histamine H1 receptor activity modulates proinflammatory cytokine production of dendritic cells through c-Rel activity . BACKGROUND : DB11320 exerts diverse effects on immune regulation through four types of histamine receptors ( HRs ) . Among them , type 1 receptor ( P35367 ) plays an important role in allergic inflammation . Dendritic cells ( DCs ) , which express at least three types of HRs , are professional antigen-presenting cells controlling the development of allergic inflammation . However , the molecular mechanisms involved in P35367 -mediated NF-ĸB signaling of DCs remain poorly defined . METHODS : Bone-marrow ( BM ) -derived DCs ( BM-DCs ) were treated with P35367 inverse agonists to interrupt basal P35367 -mediated signaling . The crosstalk of P35367 -mediated signaling and the NF-ĸB pathway was examined by NF-ĸB cellular activity using a luciferase reporter assay , NF-ĸB subunit analysis using Western blotting and P01375 -α promoter activity using chromatin immunoprecipitation . RESULTS : Blockage of P35367 signaling by inverse agonists significantly inhibited P01375 -α and P05231 production of BM-DCs . P35367 -specific agonists were able to enhance P01375 -α production , but this overexpression was significantly inhibited by NF-ĸB inhibitor . The P35367 inverse agonist ketotifen also suppressed cellular NF-ĸB activity , suggesting crosstalk between P35367 and NF-ĸB signaling in DCs . After comprehensive analysis of NF-ĸB subunits , c-Rel protein expression was significantly down-regulated in ketotifen-treated BM-DCs , which led to inhibition of the promoter activity of P01375 -α . Finally , adoptive transfer of the ketotifen-treated BM-DCs did not induce significant allergic airway inflammation compared to that of control cells in vivo . CONCLUSIONS : Our results suggest that c-Rel controls P35367 -mediated proinflammatory cytokine production in DCs . This study provides a potential mechanism of P35367 -mediated signaling and NF-ĸB pathway crosstalk in allergic inflammation . Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins . Botulinum neurotoxins ( BoNTs ) and tetanus neurotoxin ( TeNT ) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors P60880 , syntaxin , and vesicle-associated membrane protein ( VAMP ) /synaptobrevin . TeNT and BoNT/B , D , F , and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin . Except for BoNT/B and TeNT , they cleave unique peptide bonds , and prior work suggested that different substrate segments are required for the interaction of each toxin . Although the mode of P60880 cleavage by DB00083 and E has recently been studied in detail , the mechanism of VAMP/synaptobrevin proteolysis is fragmentary . Here , we report the determination of all substrate residues that are involved in the interaction with BoNT/B , D , and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin . For each of the toxins , three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding . These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin . Substrate segments C-terminal of the cleavage site ( P4-P4 ' ) do not play a role in the catalytic process . Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number ; however , the importance of individual positions at the cleavage sites varied for each toxin . The data show that , similar to the P60880 proteolyzing DB00083 and E , VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction , employing an exosite located N-terminal of the scissile peptide bond . Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and P60880 in neuron survival . Botulinum neurotoxins ( DB00083 -G ) act by blocking synaptic vesicle exocytosis . Whether BoNTs disrupt additional neuronal functions has not been addressed . Here we report that cleavage of syntaxin 1 by BoNT/C , and cleavage of P60880 by BoNT/E both induce degeneration of neurons . Furthermore , although P60880 cleaved by DB00083 still supports neuron survival , it has reduced capacity to tolerate additional mutations . We demonstrate that syntaxin 1 and P60880 cooperate as SNARE proteins to support neuron survival . Exogenous expression of other homologous SNARE proteins , syntaxin 2/3/4 and O00161 , which are resistant to BoNT/C and E in neurons , can substitute syntaxin 1/ P60880 and prevent toxin-induced neuron death . Finally , we find that neuronal death is due to blockage of plasma membrane recycling processes that utilize syntaxin 1/ P60880 , independent of synaptic vesicle exocytosis . These findings establish neuronal cytotoxicity for BoNTs and reveal syntaxin 1/ P60880 as the ubiquitous and essential SNARE proteins mediating multiple fusion events on neuronal plasma membranes . Self-assembled peptide monolayers as a toxin sensing mechanism within arrayed microchannels . A sensor for the lethal bacterial enzyme , botulinum neurotoxin type A ( DB00083 ) , was developed using self-assembled monolayers ( SAMs ) . SAMs consisting of an immobilized synthetic peptide that mimicked the toxin 's in vivo P60880 protein substrate were formed on Au and interfaced with arrayed microfluidic channels . Efforts to optimize DB00118 composition and assay conditions for greatest reaction efficiency and sensitivity are described in detail . Channel design provided facile fluid manipulation , sample incubation , analyte concentration , and fluorescence detection all within a single microfluidic channel , thus avoiding sample transfer and loss . Peptide SAMs were exposed to varying concentrations of DB00083 or its catalytic light chain ( ALC ) , resulting in enzymatic cleavage of the peptide substrate from the surface . Fluorescence detection was achieved down to 20 pg/mL ALC and 3 pg/mL DB00083 in 3 h . Toxin sensing was also accomplished in vegetable soup , demonstrating practicality of the method . The modular design of this microfluidic DB00118 platform allows for extension to sensing other toxins that operate via enzymatic cleavage , such as the remaining BoNT serotypes B-G , anthrax , and tetanus toxin . Accelerated intoxication of GABAergic synapses by botulinum neurotoxin A disinhibits stem cell-derived neuron networks prior to network silencing . Botulinum neurotoxins ( BoNTs ) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses , preventing neurotransmitter release . Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo . Consequently , the dynamic effects of intoxication on synaptic behaviors are not well-understood . We have reported that mouse embryonic stem cell-derived neurons ( ESNs ) are highly sensitive to BoNT based on molecular readouts of intoxication . Here we study the time-dependent changes in synapse- and network-level behaviors following addition of DB00083 to spontaneously active networks of glutamatergic and GABAergic ESNs . Whole-cell patch-clamp recordings indicated that DB00083 rapidly blocked synaptic neurotransmission , confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism . Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses , which was consistent with the selective accumulation of cleaved P60880 at Q99259 (+) pre-synaptic terminals at early timepoints . Different latencies of intoxication resulted in complex network responses to DB00083 addition , involving rapid disinhibition of stochastic firing followed by network silencing . Synaptic activity was found to be highly sensitive to P60880 cleavage , reflecting the functional consequences of the localized cleavage of the small subpopulation of P60880 that is engaged in neurotransmitter release in the nerve terminal . Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT . Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state : Evidence for noncanonical zinc protease activity . Clostridium botulinum neurotoxins ( BoNTs ) , the most potent toxins known , disrupt neurotransmission through proteolysis of proteins involved in neuroexocytosis . The light chains of BoNTs are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates . We have determined the crystal structure of the protease domain from BoNT serotype A ( DB00083 ) . The structure reveals a homodimer in a product-bound state , with loop F242-V257 from each monomer deeply buried in its partner 's catalytic site . The loop , which acts as a substrate , is oriented in reverse of the canonical direction for other zinc proteases . The Y249-Y250 peptide bond of the substrate loop is hydrolyzed , leaving the Y249 product carboxylate coordinated to the catalytic zinc . From the crystal structure of the DB00083 protease , detailed models of noncanonical binding and proteolysis can be derived which we propose are also consistent with DB00083 binding and proteolysis of natural substrate synaptosome-associated protein of 25 kDa ( P60880 ) . The proposed DB00083 substrate-binding mode and catalytic mechanism are markedly different from those previously proposed for the BoNT serotype B . Desmopressin ( DB00035 ) induces NO production in human endothelial cells via V2 receptor- and DB02527 -mediated signaling . The hemostatic agent desmopressin ( DB00035 ) also has strong vasodilatory effects . DB00035 is a selective agonist for the vasopressin V2 receptor ( P30518 ) , which is coupled to DB02527 -dependent signaling . DB00035 -induced vasodilation may be due to endothelial NO synthase ( P29474 ) activation . This hypothesis implies DB02527 -mediated P29474 activation . It also implies wide extrarenal , endothelial P30518 expression . We show that in human umbilical vein endothelial cells ( HUVECs ) the DB02527 -raising agents forskolin and epinephrine increase NO production , as measured by a l-NMMA-inhibitable rise in cellular cGMP content . They also increase P29474 enzymatic activity , in a partly calcium-independent manner . DB02527 -mediated P29474 activation is associated with phosphorylation of residue Ser1177 , in a phosphatidyl inositol 3-kinase ( PI3K ) -independent manner . HUVECs do not express P30518 . However , after heterologous P30518 expression , DB00035 induces DB02527 -dependent P29474 activation via Ser1177 phosphorylation . We have previously found P30518 expression in cultured lung endothelial cells . By real time quantitative RT-PCR , we now find a wide P30518 distribution notably in heart , lung and skeletal muscle . These results indicate that DB00035 and other DB02527 -raising agents can activate P29474 via PI3K-independent Ser1177 phosphorylation in human endothelial cells . This mechanism most likely accounts for DB00035 -induced vasodilation . Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B . Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT ( botulinum neurotoxin ) . The DB00083 complex is widely used because of its longer-lasting benefits ; also , autonomic side-effects are more often reported for BoNT/B . To establish if this distinct effect of BoNT/B could be exploited therapeutically , DB00083 was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action . The advantageous protease and translocation domain of DB00083 were recombinantly combined with the acceptor-binding moiety of type B [ H(C)/B ( C-terminal half of BoNT/B heavy chain ) ] , creating the chimaera AB . This purified protein bound the BoNT/B acceptor , displayed enhanced capability relative to type A for intraneuronally delivering its protease , cleaved P60880 ( synaptosome-associated protein of 25 kDa ) and induced a more prolonged neuromuscular paralysis than DB00083 in mice . The BA chimaera , generated by substituting H(C)/A ( C-terminal half of DB00083 heavy chain ) into BoNT/B , exhibited an extremely high specific activity , delivered the BoNT/B protease via the DB00083 acceptor into neurons , or fibroblast-like synoviocytes that lack P60880 , cleaving the requisite isoforms of VAMP ( vesicle-associated membrane protein ) . Both chimaeras inhibited neurotransmission in murine bladder smooth muscle . BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the DB00083 -acceptor and utilising VAMP , but not P60880 , in exocytosis . Acute and chronic effects of botulinum neurotoxin a on the mammalian neuromuscular junction . INTRODUCTION : Botulinum neurotoxin A ( DB00083 ) cleaves P60880 and inhibits acetylcholine ( ACh ) release at the neuromuscular junctions ( NMJ ) to cause neuroparalysis . Previous reports indicate a dyssynchrony between the inhibitory effect of DB00083 on ACh release and P60880 cleavage . METHODS : We tested the in vitro ( acute ; 90 min ) and in vivo ( chronic ; 12 h ) effects of DB00083 on stimulus-evoked ACh release ( SEAR ) , twitch tension , and P60880 cleavage in isolated extensor digitorum longus ( Q9Y5X9 ) nerve-muscle preparations ( NMP ) . RESULTS : In vitro or in vivo DB00083 poisoning inhibited SEAR and twitch tension . Conversely , P60880 cleavage and inhibition of spontaneous release frequency were observed only in NMP poisoned with DB00083 in vivo . Moreover , chronic treatment of DB00083 inhibited ionomycin stimulated Ca(2+) signals in Neuro 2a cells . CONCLUSIONS : These results demonstrate that the inhibition of SEAR precedes P60880 cleavage and suggest involvement of a more complex mechanism for the inhibitory effect of DB00083 at the NMJ . Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins : the extent and duration are dictated by the sites of P60880 truncation . Nerve sprouts emerge from motor nerve terminals following blockade of exo-endocytosis for more than 3 days by botulinum neurotoxin ( BoNT ) , and form functional synapses , albeit temporary . Upon restoration of synaptic activity to the parent terminal 7 and 90 days after exposure to BoNT/F or A respectively , a concomitant retraction of the outgrowths was observed . BoNT/E caused short-term neuroparalysis , and dramatically accelerated the recovery of DB00083 -paralyzed muscle by further truncation of P60880 and its replenishment with functional full-length SNARE . The removal of 9 C-terminal residues from P60880 by DB00083 leads to persistence of the inhibitory product due to the formation of a nonproductive SNARE complex(es) at release sites , whereas deletion of a further 17 amino acids permits replenishment and a speedy recovery . Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of P60880 . Botulinum neurotoxin serotypes A and E ( DB00083 and BoNT/E ) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein , P60880 . For each BoNT serotype , cleavage of P60880 results in the loss of intact protein , the production of an N-terminal truncated protein , and the generation of a small C-terminal peptide . Peptides that mimic the C-terminal fragments of P60880 following DB00083 or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells . Similarly , the N-terminal-truncated P60880 resulting from DB00083 or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems . With one exception , however , the inhibitory action of truncated P60880 has not been demonstrated at a well-defined cholinergic synapse . The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal DB00083 - or BoNT/E-truncated P60880 in two different neuronal systems : cholinergically coupled Aplysia neurons and rat hippocampal cell cultures . Both truncated P60880 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells . These results suggest that truncated P60880 can compete with endogenous P60880 for binding with other SNARE proteins involved in transmitter release , thus inhibiting neurotransmitter exocytosis . Recombinant holotoxoid vaccine against botulism . The botulinum neurotoxins ( BoNT ) are the most toxic proteins for humans and designated " Category A Select Agents. " The current vaccine against botulism is in limited supply , and there is a need to develop new vaccine strategies . A recombinant DB00083 toxoid was produced in Clostridium botulinum that contained a double amino acid substitution , R363A Y365F ( termed DB00083 (RYM) ) . DB00083 (RYM) was noncatalytic for P60880 and nontoxic for mice . Immunization with DB00083 (RYM) protected mice from challenge at levels that were similar to chemically inactivated DB00083 toxoid . DB00083 (RYM) elicited an immune response against the light-chain and heavy-chain components of the toxin . Neutralizing anti- DB00083 (RYM) sera blocked BoNT toxicity in primary cortical neurons and blocked ganglioside binding by the heavy chain . DB00083 (RYM) represents a viable vaccine candidate for a holotoxoid against botulism . Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay . Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E ( DB00083 and BoNT/E ) . As detailed in that article , the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein ( Q9ULX5 ) and green fluorescent protein ( GFP ) moieties linked via residues 134-206 of P60880 ( synaptosome-associated protein of 25kDa ) , the protein substrate for DB00083 and BoNT/E . Before cleavage of this recombinant substrate , the polarization observed for the GFP emission , excited near the absorption maximum of the Q9ULX5 , is very low due to depolarization following energy transfer from Q9ULX5 to GFP . After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance , the polarization is high due to observation of the emission only from directly excited GFP . This change in fluorescence polarization allows an assay , termed DARET ( depolarization after resonance energy transfer ) , that is robust and sensitive . In this article , we characterize the spectroscopic parameters of the system before and after substrate cleavage , including excitation and emission spectra , polarizations , and lifetimes . Mastoparan-7 rescues botulinum toxin-A poisoned neurons in a mouse spinal cord cell culture model . Botulinum neurotoxin serotype A ( DB00083 ) is the most potent poison of biological origin known to mankind . The toxicity of DB00083 is due to the inhibition of neurotransmission at cholinergic synapses ; this is responsible for the symptom of flaccid paralysis at peripheral neuromuscular junctions . At a molecular level , the DB00083 effect is due to its inhibition of stimulated acetylcholine ( ACh ) release from presynaptic nerve terminals . Currently , there is no antidote available to rescue DB00083 -poisoned synapses . Here , we report an example of rescuing botulinum-poisoned cultured mouse spinal cord neurons by treatment with Mastoparan-7 ( Mas-7 ) , which is known to be a phospholipase A2 activator compound . Mas-7 , a naturally occurring bee venom peptide , was delivered to botulinum-poisoned neurons via a drug delivery vehicle ( DDV ) construct prepared using the recombinant non-toxic heavy chain ( HC ) fragment of DB00083 itself . In this method , the DB00083 HC component in the DDV served as a neuron specific drug targeting molecule . We found that Mas-7 delivered into DB00083 intoxicated spinal cord cells restored over 40 % their property of stimulated neurotransmitter release . Rescue from cell poisoning did not occur from inhibition of the endopeptidase activity of DB00083 light chain ( LC ) against its well-known substrate , P60880 that is mechanistically involved in the cholinergic neuroexocytosis process . Rather , Mas-7 induced a physiological host response apparently unrelated to P60880 , but linked to the phospholipase-mediated signal transduction pathway . Probing DB00083 protease exosites : implications for inhibitor design and light chain longevity . Botulinum neurotoxin serotype A ( DB00083 ) is one of the most lethal toxins known . Its extreme toxicity is due to its light chain ( LC ) , a zinc protease that cleaves P60880 , a synaptosome-associated protein , leading to the inhibition of neuronal activity . Studies on DB00083 LC have revealed that two regions , termed exosites , can play an important role in BoNT catalytic activity . A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of P60880 cleavage and the design of inhibitors . Herein , based on the crystallographic structure of DB00083 LC complexed with its substrate , we designed an α-exosite binding probe . Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC . These data help delineate why α-exosite binding is needed for P60880 cleavage and also provide new insights into the extended lifetime observed for DB00083 LC in vivo . 8-Hydroxyquinoline and hydroxamic acid inhibitors of botulinum neurotoxin DB00083 . We describe here the state of the art of certain aspects concerning potential small molecule therapy directed toward botulism , by inhibition of the zinc-protease containing light chain ( LC ) of botulinum neurotoxin DB00083 from the anaerobic bacillus Clostridium botulinum . Botulinum neurotoxins ( BoNTs ) are comprised of eight serologically-distinct proteins ( A - H ) , several of which are further divided , such as DB00083 which has five subtypes . The BoNTs are the most toxic substances known to mankind , causing a form of flaccid paralysis that can be rapid and is often lethal . DB00083 is comprised of a ~100 kDa heavy chain ( HC ) attached via a single disulfide DB00151 - DB00151 bond to a ~50 kDa LC . The HC mediates transport to and uptake by presynaptic glutamatergic neurons , where the LC cleaves the protein P60880 and thus prevents vesicular trafficking and release of acetylcholine . The Zn-endoprotease activity of the LC of DB00083 is a target for the development of small molecule inhibitors of DB00083 -mediated toxicity . A variety of DB00083 LC inhibitors have been described to date and we focus here primarily on the Zn-binding 8-hydroxyquinoline structural type as well as some of the previously-described hydroxamic acids . DB00435 suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of IkappaB . The expression of inducible nitric oxide synthase ( P35228 ) is a critical factor in both normal physiological functions and the pathogenesis of disease . This study was undertaken to determine the molecular mechanism by which nitric oxide ( NO ) exerts negative feedback regulation on P35228 gene expression . Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as P35228 mRNA and protein levels , which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D,L-penicillamine ( P60880 ) and V-PYRRO/NO . This effect of P60880 was inhibited when NO was scavenged using red blood cells . Pretreatment with oxidized P60880 , 8-Br-cGMP , NO2- , or NO3- did not suppress the cytokine-induced NO production . Moreover , LPS/ P01579 -stimulated RAW264.7 cells , which produce endogenous NO , expressed lower levels of P35228 , IL-1beta , P05231 and P01375 mRNAs , without changes in their mRNA half-lives , than those in the presence of the P35228 inhibitor NG-monomethyl-L-arginine . The P35228 gene transcription rate exhibited an 18-fold increase after cytokine stimulation , which was significantly inhibited by P60880 pretreatment . P60880 also blocked cytokine- induced increase in NF-kappaB activation , P35228 promoter activity , nuclear translocation of cytosolic NF-kappaB p65 subunit , and P25963 degradation , which correlated with its inhibitory effect on phosphorylation and ubiquitination of IkappaB . These data indicate that NO down-regulates P35228 gene expression and NO production by inhibiting the post-translational processes of P25963 thereby preventing NF-kappaB activation . These results identify a novel negative feedback mechanism whereby NO down-regulates P35228 gene expression . Adeno-associated virus transfer of a gene encoding P60880 resistant to botulinum toxin A attenuates neuromuscular paralysis associated with botulism . Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases , but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons , ambiguities in the identity of gene(s) implicated , and a paucity of means to quantify any physiological improvement . Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A ( DB00083 ) as a prototype disease . Furthermore , because human botulism , occasionally fatal , causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin 's P60880 ( S25 ) -cleaving protease , development of a genetic approach could lead to a potential treatment for this debilitating disease . Adeno-associated viral delivery of a cleavage-resistant S25 gene ( S25-R198T ) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by DB00083 of evoked catecholamine release . Evaluation in vivo , by administering this virus into rat spinal cord before injecting DB00083 , showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission . A similar , although smaller , protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus . Such therapy also curtailed nerve sprouting normally induced by DB00083 . This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons . DB00741 is a suppressor of apoptosis in bovine corpus luteum . Glucocorticoid ( GC ) acts as a modulator of physiological functions in several organs . In the present study , we examined whether GC suppresses luteolysis in bovine corpus luteum ( CL ) . DB00741 ( an active GC ) reduced the mRNA expression of caspase 8 ( Q14790 ) and caspase 3 ( P42574 ) and reduced the enzymatic activity of P42574 and cell death induced by tumor necrosis factor ( P01375 ) and interferon gamma ( P01579 ) in cultured bovine luteal cells . mRNAs and proteins of GC receptor ( P04150 ) , 11beta-hydroxysteroid dehydrogenase type 1 ( P28845 ) , and P80365 were expressed in CL throughout the estrous cycle . Moreover , the protein expression and the enzymatic activity of P28845 were high at the early and the midluteal stages compared to the regressed luteal stage . These results suggest that cortisol suppresses P01375 - P01579 -induced apoptosis in vitro by reducing apoptosis signals via Q14790 and P42574 in bovine CL and that the local increase in cortisol production resulting from increased P28845 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells . Is nitric oxide involved in P41595 receptor-mediated contraction in the rat stomach fundus ? Although contraction of the rat stomach fundus by 5-HT is known to be mediated by the P41595 receptor , the second messenger pathways involved in this response remain unclear . Since nitric oxide ( NO ) has been associated with contraction of certain gastrointestinal smooth muscle , the purpose of this study was to determine if NO is involved in 5-HT-induced contraction in the rat stomach fundus . The arginine analogs L-NAME and L-NMMA , at a concentration ( 100 microM ) established to inhibit NO synthase in the rat stomach fundus by inhibiting depolarization-induced relaxation in this tissue , had no effect on 5-HT contraction . Furthermore , the NO donors sodium nitroprusside and P60880 did not contract rat stomach fundus under basal tone , whereas 5-HT was a potent contractile agonist . These data do not support a role for NO in P41595 receptor-mediated contraction in the rat stomach fundus . Protective effects of metallothionein on isoniazid and rifampicin-induced hepatotoxicity in mice . Isoniazid ( DB00951 ) and DB01045 ( RFP ) are widely used in the world for the treatment of tuberculosis , but the hepatotoxicity is a major concern during clinical therapy . Previous studies showed that these drugs induced oxidative stress in liver , and several antioxidants abated this effect . Metallothionein ( MT ) , a member of cysteine-rich protein , has been proposed as a potent antioxidant . This study attempts to determine whether endogenous expression of MT protects against DB00951 and RFP-induced hepatic oxidative stress in mice . Wild type ( MT+/+ ) and MT-null ( MT-/- ) mice were treated intragastrically with DB00951 ( 150 mg/kg ) , RFP ( 300 mg/kg ) , or the combination ( 150 mg/kg DB00951 +300 mg/kg RFP ) for 21 days . The results showed that MT-/- mice were more sensitive than MT+/+ mice to DB00951 and RFP-induced hepatic injuries as evidenced by hepatic histopathological alterations , increased serum Q9NRA2 levels and liver index , and hepatic oxidative stress as evidenced by the increase of MDA production and the change of liver antioxidant status . Furthermore , DB00951 increased the protein expression of hepatic P05181 and DB00951 /RFP ( alone or in combination ) decreased the expression of hepatic P05177 . These findings clearly demonstrate that basal MT provides protection against DB00951 and RFP-induced toxicity in hepatocytes . The P05181 and P05177 were involved in the pathogenesis of DB00951 and RFP-induced hepatotoxicity . Gender difference in the activity but not expression of estrogen receptors alpha and beta in human lung adenocarcinoma cells . The higher frequency of lung adenocarcinoma in women smokers than in men smokers suggests a role for gender-dependent factors in the etiology of lung cancer . We evaluated estrogen receptor ( ER ) alpha and beta expression and activity in human lung adenocarcinoma cell lines and normal lung fibroblasts . Q8N1N2 -length ERalpha and ERbeta proteins were expressed in all cell lines with higher ERbeta than ERalpha . Although estradiol ( E(2) ) binding was similar , E(2) stimulated proliferation only in cells from females , and this response was inhibited by anti-estrogens 4-hydroxytamoxifen ( DB04468 ) and DB00947 . In contrast , E(2) did not stimulate replication of lung adenocarcinoma cells from males and DB04468 or ICI did not block cell proliferation . Similarly , transcription of an estrogen response element-driven reporter gene was stimulated by E(2) in lung adenocarcinoma cells from females , but not males . P06401 ( PR ) expression was increased by E(2) in two out of five adenocarcinoma cell lines from females , but none from males . E(2) decreased P12830 protein expression in some of the cell lines from females , as it did in MCF-7 breast cancer cells , but not in the cell lines from males . Thus , ERalpha and ERbeta expression does not correlate with the effect of ER ligands on cellular activities in lung adenocarcinoma cells . On the other hand , coactivator Q15648 expression was higher in lung adenocarcinoma cells from females versus males and higher in adenocarcinoma cells than in normal human bronchial epithelial cells . Q15648 and other ER coregulators may contribute to differences in estrogen responsiveness between lung adenocarcinoma cells in females and males . Peptide inhibitors of botulinum neurotoxin by mRNA display . Botulinum neurotoxins ( BoNTs ) are extremely toxic . The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion . Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs . Toward that goal , we produced a synthetic cDNA for the expression and purification of the metalloprotease of DB00083 in Escherichia coli as a biotin-ubiquitin fusion protein , and constructed a combinatorial peptide library to screen for DB00083 light chain inhibitors using mRNA display . A protease assay was developed using immobilized intact P60880 as the substrate . The new peptide inhibitors showed a 10-fold increase in affinity to DB00083 light chain than the parent peptide . Interestingly , the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues . The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs . Molecular targets of botulinum toxin at the mammalian neuromuscular junction . The molecular targets of botulinum neurotoxins ( BoNTs ) are SNARE ( soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor ) proteins necessary for neurotransmitter release . BoNT are powerful therapeutic agents in the treatment of numerous neurological disorders . The goals of this study were to ( 1 ) assess toxin diffusion by measuring substrate cleavage in adjacent and distant muscles , and ( 2 ) characterize the clinical course using SNARE protein chemistry . A small volume of DB00083 was injected unilaterally into the mouse gastrocnemius muscle . Motor impairment was limited to the toxin-treated limb . No systemic illness or deaths occurred . At five time points , a subset of mice were killed , and muscles from both hindlimbs , and the diaphragm , were collected . Protein samples were examined for changes in P60880 ( synaptosomal-associated protein of Mr = 25 kDa ) using immunochemistry . P60880 cleavage product was noted in the toxin-treated limb as early as 1 day postinjection and continued through day 28 . Onset and peak levels of substrate cleavage corresponded to the onset and peak clinical response . Cleavage was observed in adjacent and distant muscles , demonstrating that substrate cleavage is a sensitive indicator of toxin diffusion . Significant increases in full-length P60880 and vesicle-associated membrane protein II were evident early in the impaired limb and continued through day 28 . The increased SNARE protein most likely originates from nerve terminal sprouts . A high-density single-nucleotide polymorphism screen of 23 candidate genes in attention deficit hyperactivity disorder : suggesting multiple susceptibility genes among Chinese Han population . Attention deficit hyperactivity disorder ( ADHD ) is a common childhood-onset behavioral disorder with a definite genetic component . The search for genes predisposing to ADHD has focused on genes involved in the regulation of monoamine systems . In this study , we emphasized genes that underlie various aspects of dopamine , norepinephrine and serotonin neurotransmissions and performed a comprehensive association analysis by screening with 245 single-nucleotide polymorphisms ( SNPs ) of 23 candidate genes in a sample of Chinese Han descent . A total of 182 DSM-IV ADHD children and 184 healthy controls were genotyped and analyzed with an average density of one SNP every 6.1 kb . Both single-SNP and multi-marker haplotype analyses were implemented to exploit association signal for ADHD and its diagnostic subtypes . Empirical P-values were derived on the basis of 5000 permutations to evaluate gene-wide statistical significance . P21397 yielded highly suggestive evidence of association ( empirical P < 0.01 , OR=1.94 ) with ADHD . For inattentive ADHD , P21397 , DDC and P08247 showed suggestive evidence of association ( empirical P < 0.05 ) . P18825 achieved suggestive significance ( empirical P < 0.05 ) for ADHD combined type . Additionally , for six genes ( P60880 , NET1 , P09172 , P43681 , P35462 and P21579 ) we detected one or more SNPs with nominal P-values </= 0.05 . This study has identified several genes as promising susceptibility loci for ADHD . Replication efforts and further investigations remain necessary to provide definite proof of association . Long-distance retrograde effects of botulinum neurotoxin A . Botulinum neurotoxins ( designated DB00083 -BoNT/G ) are bacterial enzymes that block neurotransmitter release by cleaving essential components of the vesicle fusion machinery . DB00083 , which cleaves P60880 ( synaptosomal-associated protein of 25 kDa ) , is extensively exploited in clinical medicine to treat neuromuscular pathologies , facial wrinkles , and various types of pain . It is widely assumed that DB00083 remains at the synaptic terminal and its effects are confined to the injection site . Here we demonstrate that catalytically active DB00083 is retrogradely transported by central neurons and motoneurons and is then transcytosed to afferent synapses , in which it cleaves P60880 . P60880 cleavage by DB00083 was observed in the contralateral hemisphere after unilateral DB00083 delivery to the hippocampus . Appearance of cleaved P60880 resulted in blockade of hippocampal activity in the untreated hemisphere . Injections of DB00083 into the optic tectum led to the appearance of DB00083 -truncated P60880 in synaptic terminals within the retina . Cleaved P60880 also appeared in the facial nucleus after injection of the toxin into rat whisker muscles . Experiments excluded passive spread of the toxin and demonstrated axonal migration and neuronal transcytosis of DB00083 . These findings reveal a novel pathway of DB00083 trafficking in neurons and have important implications for the clinical uses of this neurotoxin . Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates . Multi-domain bacterial protein toxins are being explored as potential carriers for targeted delivery of biomolecules . Previous approaches employing isolated receptor binding subunits disallow entry into the cytosol . Strategies in which catalytic domains are replaced with cargo molecules are presumably inefficient due to co-operation of domains during the endosomal translocation step . Here , we characterize a novel transport vehicle in which cargo proteins are attached to the amino terminus of the full-length botulinum neurotoxin type D ( BoNT/D ) . The intrinsic enzymatic activity of the neurotoxin allowed quantification of the efficacy of cargo delivery to the cytosol . P00374 and BoNT type A ( DB00083 ) light chain ( LC ) were efficiently conveyed into the cytosol , whereas attachment of firefly luciferase or green fluorescent protein drastically reduced the toxicity . Luciferase and DB00083 LC retained their catalytic activity as evidenced by luciferin conversion or P60880 hydrolysis in the cytosol of synaptosomes , respectively . Conformationally stabilized dihydrofolate reductase as cargo considerably decreased the toxicity indicative for the requirement of partial unfolding of cargo protein and catalytic domain as prerequisite for efficient translocation across the endosomal membrane . Thus , enzymatically inactive clostridial neurotoxins may serve as effective , safe carriers for delivering proteins in functionally active form to the cytosol of neurones . Widespread sequence variations in P23763 across vertebrates suggest a potential selective pressure from botulinum neurotoxins . Botulinum neurotoxins ( DB00083 -G ) , the most potent toxins known , act by cleaving three SNARE proteins required for synaptic vesicle exocytosis . Previous studies on BoNTs have generally utilized the major SNARE homologues expressed in brain ( P63027 , syntaxin 1 , and P60880 ) . However , BoNTs target peripheral motor neurons and cause death by paralyzing respiratory muscles such as the diaphragm . Here we report that P23763 , but not P63027 , is the SNARE homologue predominantly expressed in adult rodent diaphragm motor nerve terminals and in differentiated human motor neurons . In contrast to the highly conserved P63027 , BoNT-resistant variations in P23763 are widespread across vertebrates . In particular , we identified a polymorphism at position 48 of P23763 in rats , which renders P23763 either resistant ( I48 ) or sensitive ( M48 ) to BoNT/D . Taking advantage of this finding , we showed that rat diaphragms with I48 in P23763 are insensitive to BoNT/D compared to rat diaphragms with M48 in P23763 . This unique intra-species comparison establishes P23763 as a physiological toxin target in diaphragm motor nerve terminals , and demonstrates that the resistance of P23763 to BoNTs can underlie the insensitivity of a species to members of BoNTs . Consistently , human P23763 contains I48 , which may explain why humans are insensitive to BoNT/D . Finally , we report that residue 48 of P23763 varies frequently between M and I across seventeen closely related primate species , suggesting a potential selective pressure from members of BoNTs for resistance in vertebrates . A structural perspective of the sequence variability within botulinum neurotoxin subtypes A1-A4 . Botulinum neurotoxin ( BoNT ) is a category A toxin that has been classified within seven serotypes , designated A-G . Recently , it has been discovered that sequence variability occurs in BoNTs produced by serotype A ( DB00083 ) variant strains , designated as subtypes A1 and A2 , which have significantly different antibody-binding properties . We have therefore made efforts to understand at the molecular level the diversity and its effects on the biological actions of the toxin , including receptor binding , substrate recognition , and catalysis . We provide the results of these studies , including the analysis of two newly sequenced DB00083 variants , Loch Maree ( A3 ) and 657Ba ( A4 ) , and their comparison to A1 and A2 . Using sequence analysis , available functional data , molecular modeling , and comparison of models with the crystal structures of BoNT/A1 and the light chain of BoNT/A2 , we conclude that these sequence differences within subtypes will impact development of broad-spectrum antibody and small ligand therapeutics , and suggest dissimilarities in binding affinity and cleavage efficiency of the P60880 substrate . In particular , sequence variation in subtypes BoNT/A3 and BoNT/A4 will likely effect alpha-exosite and S1 ' subsite recognition , respectively . P01308 / P05019 signaling pathways enhances tumor cell invasion through bisecting GlcNAc N-glycans modulation . an interplay with P12830 . Changes in glycosylation are considered a hallmark of cancer , and one of the key targets of glycosylation modifications is P12830 . We and others have previously demonstrated that P12830 has a role in the regulation of bisecting GlcNAc N-glycans expression , remaining to be determined the P12830 -dependent signaling pathway involved in this N-glycans expression regulation . In this study , we analysed the impact of P12830 expression in the activation profile of receptor tyrosine kinases such as insulin receptor ( IR ) and P08069 ( IGF-IR ) . We demonstrated that exogenous P12830 expression inhibits IR , IGF-IR and P29323 1/2 phosphorylation . Stimulation with insulin and P05019 in MDA-MD-435 cancer cells overexpressing P12830 induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on P12830 cellular localization . Concomitantly , IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability . Altogether , these results demonstrate an interplay between P12830 and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression , with important effects in the modulation of epithelial characteristics and tumor cell invasion . Here we provide new insights into the role that P01308 / P05019 signaling play during cancer progression through glycosylation modifications . 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 . Chapter 3 : Molecular basis for the therapeutic effectiveness of botulinum neurotoxin type A . The utility of botulinum neurotoxin type A ( DB00083 ) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission . Specific , high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 ( SV2 ) by the heavy chain ( HC ) of DB00083 confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis . Upon vesicle acidification , the HC forms a channel for transmembrane transfer of the light chain to the cytosol , as observed by single channel recordings . The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates P60880 , thereby blocking exocytotic release of transmitters , a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion . A di-leucine motif in DB00083 light chain stabilizes this protease , contributing to its longevity inside nerves . The ubiquity of SV2 and P60880 has prompted re-evaluation of the nerve types susceptible to DB00083 . In urology , there is emerging evidence that DB00083 blocks neuropeptide release from afferent nerves , exocytosis of acetylcholine and purines from efferent nerves , and possibly DB00171 release from the urothelium . Suppression by DB00083 of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms . DB00435 induces apoptosis in GM- P04141 -treated eosinophils via caspase-6-dependent lamin and DNA fragmentation . Asthma is characterized by accumulation of eosinophils in the lungs and delayed apoptosis may be one mechanism leading to eosinophilia . DB00435 ( NO ) , present in inflamed lungs , has been shown to possess both anti- and proeosinophilic properties . We previously showed that NO induces apoptosis in the presence of survival prolonging cytokine P05113 in human eosinophils . In the present study , we examined the intracellular mechanisms of NO-induced apoptosis in granulocyte macrophage-colony stimulating factor ( GM- P04141 ) -treated eosinophils concentrating on the role of caspases and calpains . Eosinophils were isolated from human blood and apoptosis was determined by relative DNA fragmentation assay , morphological analysis and/or Annexin-V FITC assay . We showed that NO-donor S-nitroso-N-acetyl-d,l-penicillamine ( P60880 ) induced apoptosis in GM- P04141 -treated eosinophils . P60880 -induced DNA fragmentation was totally prevented by an inhibitor of caspase-6 ( Z-VEID-FMK ) . Decreased levels of caspase-6 proenzyme and increased amounts of cleaved lamin A/C in P60880 -treated cells indicated activation of caspase-6 . Furthermore , P60880 -induced lamin A/C and B fragmentation was totally abolished by an inhibitor of caspase-6 . According to our results , caspase-6 mediates lamin and DNA fragmentation also in spontaneously dying eosinophils . Inhibitor of calpains prevented most of DNA fragmentation related to spontaneous apoptosis but had no effect in eosinophils undergoing NO-induced apoptosis . In the present study we showed that caspase-6 is essential for the executive phase involving lamin and DNA fragmentation in both NO-induced and spontaneous eosinophil apoptosis . However , differences in the involvement of calpains suggest that the intracellular signalling in NO-induced apoptosis has specific features at the level of proteases . This study demonstrates new mechanisms for NO-induced and spontaneous apoptosis in human eosinophils . Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity . The light chain of botulinum neurotoxin A ( DB00083 -LC ) is a zinc-metalloprotease that requires two extended exosites for optimal substrate binding and recognition of its intracellular target P60880 . P27918 and YFP connected through P60880 peptide ( 141-206 ) containing both exosites ( CsY ) has been used in a FRET-based assay for DB00083 . To further improve the FRET efficiency in this DB00083 substrate for in vitro high-throughput assays , we explored the feasibility of enhancing the capture of P27918 emission by doubling the number of YFP acceptors . In comparison to CsY , the tandem fluorescence substrates CsYY and YsCsY enhanced the ratiometric fluorescence signal between YFP and P27918 . YsCsY , containing two substrate sites , offered the greatest fluorometric change upon toxin-catalyzed cleavage . In addition to known approaches for enhancing fluorescence yield through various mutations , this alternative tandem substrate approach can boost the FRET signal and is particularly useful for substrates requiring extensive exosite recognition for specificity . P06401 level as a predictor of response to megestrol acetate in advanced breast cancer : a retrospective study . DB00351 ( 160 mg/day ) produced a response rate of 44 % in a retrospective series of 39 evaluable patients with advanced breast cancer . The estrogen-receptor ( ER ) level was greater than 10 fmols/mg of protein in 28 patients , and the progesterone-receptor ( PR ) level was greater than 10 fmols/mg of protein in 26 patients . ER and PR levels , age , and disease-free interval were analyzed for their relationship to response . The PR was the single best predictor of response to megestrol acetate ; the addition of ER added 2 % to the predictive accuracy rate of PR alone . Therapeutic effectiveness of botulinum neurotoxin A : potent blockade of autonomic transmission by targeted cleavage of only the pertinent P60880 . In search of a basis for the impressive potency of an endoprotease that cleaves P60880 , botulinum neurotoxin type A ( DB00083 ) , in treating numerous diseases due to hyper-active autonomic nerves , truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons . DB05232 -sensitive spontaneous cholinergic neurotransmission was blocked > 80 % by 1 pM DB00083 despite cleaving < 20 % of the P60880 . A maximum cleavage of ∼60 % P60880 could be achieved with > 1 nM DB00083 , despite an absence of non-cleavable P60880 in the detergent-solubilised neurons . In contrast , BoNT/E ( 100 nM ) truncated nearly all the P60880 in the intact cells , but was unable to block neurotransmission at low concentrations like DB00083 . Chimeras created by inserting the acceptor-binding HC domain of DB00083 into BoNT/E still cleaved all the P60880 , indicating ubiquitous expression of DB00083 acceptors . Accordingly , SV2 and P60880 were found to be co-expressed and broadly co-localised in neurons , but absent from non-neuronal cells . On the other hand , partial cleavage by the DB00083 protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B . Moreover , limited cleavage of P60880 was conferred onto the protease from BoNT/E when fused to the N-terminus of DB00083 . Thus , the DB00083 protease is uniquely well-adapted for selectively inactivating the P60880 directly involved in neurotransmission ; this together with the toxin 's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic . Prevalence of borreliosis , anaplasmosis , ehrlichiosis and Dirofilaria immitis in dogs and vectors in Voronezh Reserve ( Russia ) . Most of the dogs studied for the prevalence of CVBD have previously received acaricidal and insecticidal treatments . In the present work , a very specific population of dogs ( Group 1 ) that had never been treated against ticks and mosquitoes was studied . Moreover , the territory occupied by this population has also never been treated , because it is a protected area -- Voronezh Natural Reserve . Canine patients from veterinary clinics ( Group 2 ) that had been treated against VBD vectors were studied for comparison . Eighty-two dogs ( Group 1 ) were enrolled in June , 2008 . Blood samples were tested using the IDEXX P60880 (®) 4Dx(®) test . A specific heartworm antigen was detected in 12.2 % samples . The seroprevalence for Anaplasma phagocytophilum was found to be 34.1 % . The antibodies to Borrelia P13671 peptide and to Ehrlichia canis were detected in 2.4 % of the samples . Almost all dogs with infections had no clinical signs . Only 3 mixed-infected dogs showed non-specific clinical signs . During the tick season , 358 Ixodes ricinus were collected ; the prevalence of Borrelia burgdorferi s.l. and Anaplasma phagocytophilum was 21.9 % and 0.6 % , respectively . Four hundred and forty dogs ( Group 2 ) were studied for comparison . Antibodies to B. burgdorferi s.l. were detected only in one dog , seroprevalence for A. phagocytophilum represented 1.1 % , no E. canis seropositive dogs were identified , and 8.2 % dogs were found infected with Dirofilaria immitis . Fifty-six percent of dogs with dirofilariosis had clinical signs . All dogs with anaplasmosis showed specific clinical signs -- fever , anemia , splenitis . Three dogs died within a few days . Primary cultures of embryonic chicken neurons for sensitive cell-based assay of botulinum neurotoxin : implications for therapeutic discovery . Botulinum toxin is an exceedingly potent inhibitor of neurotransmission across the neuromuscular junction , causing flaccid paralysis and death . The potential for misuse of this deadly poison as a bioweapon has added a greater urgency to the search for effective therapeutics . The development of sensitive and efficient cell-based assays for the evaluation of toxin antagonists is crucial to the rapid and successful identification of therapeutic compounds . The authors evaluated the sensitivity of primary cultures from 4 distinct regions of the embryonic chick nervous system to botulinum neurotoxin A ( DB00083 ) cleavage of synaptosomal-associated protein of 25 kD ( P60880 ) . Although differences in sensitivity were apparent , P60880 cleavage was detectable in neuronal cells from each of the 4 regions within 3 h at DB00083 concentrations of 1 nM or lower . Co-incubation of chick neurons with DB00083 and toxin-neutralizing antibodies inhibited P60880 cleavage , demonstrating the utility of these cultures for the assay of DB00083 antagonists . DB00435 -cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of DB00171 -sensitive K+ channels in rat hearts . DB00435 ( NO ) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries . The present work was performed to study better the NO-cGMP-protein kinase G ( PKG ) signaling pathway in the activation of both sarcolemmal and mitochondrial DB00171 -sensitive K+ ( KATP ) channels during anoxic preconditioning ( P25054 ) and final influence on reducing anoxia-reperfusion ( A/R ) -induced cardiac damage in rat hearts . The upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection were investigated . The involvement of both inducible and endothelial NO synthases ( P35228 and P29474 ) in the progression of this signaling pathway was followed . Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release , DNA strand breaks , and malondialdehyde formation as indexes of cell injury and lipid peroxidation , respectively , were investigated . The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial KATP channels opener diazoxide ( 100 microM ) , nonspecific mitochondrial KATP channels opener pinacidil ( 50 microM ) , S-nitroso-N-acetylpenicillamine ( P60880 , 300 microM ) , or beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclicmonophosphorothioate , Sp-isomer ( 10 microM ) before the A/R period . Preconditioning with P60880 significantly reduced the DNA damage . The effect was blocked by glibenclamide ( 50 microM ) , 5-hydroxydecanoate ( 100 microM ) , NG-nitro-L-arginine methyl ester ( 200 microM ) , and beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate , Rp-isomer ( 1 microM ) . The results suggest P35228 , rather than P29474 , as the major contributing NO synthase during P25054 treatment . Moreover , the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection during P25054 treatment . Antagonism of botulinum toxin type A-induced cleavage of P60880 in rat cerebral synaptosome by toosendanin . Toosendanin ( Q15631 ) , a triterpenoid derivative extracted from Chinese traditional medicine , has been demonstrated to be an effective cure for experimental botulism . This study is designed to explore its antibotulismic mechanism by Western blotting . The results showed that Q15631 incubation did not change the electrophoresis pattern and the amounts of synaptosomal-associated protein of 25 kDa ( P60880 ) , syntaxin and synaptobrevin/vesicle-associated membrane protein in rat cerebral synaptosomes , but made the synaptosomes completely resistant to botulinum neurotoxin A ( DB00083 ) -mediated cleavage of P60880 . After binding of DB00083 to synaptosomes , Q15631 still partially antagonized the toxin-mediated cleavage of P60880 . However , Q15631 -incubated synaptosomal membrane fraction did not resist the cleavage of P60880 by the light chain of DB00083 . It is suggested that the antibotulismic effect of Q15631 results from blocking the toxin 's approach to its enzymatic substrate . [ Innate resistance to thymidylate synthase inhibition after 5-fluorouracil treatment -- a rationale of combined use of cisplatin and its optimal administration dose ] . We examined the changes of the number of DB00322 MP binding sites of thymidylate thynthase ( TS-BS ) in Yoshida sarcoma after administration of DB00544 to the tumor bearing rats . We also investigated the optimal dose of DB00515 for the increase of intracellular folate level . In the group received consecutive 7-days administration of DB09327 ( U-7 group ) , total TS-BS was significantly increased compared with non-treatment group and the group received only DB09327 ( U-1 group ) . For free TS-BS , however , there was no difference despite of DB09327 administration . P04818 inhibition rate ( TSIR ) was , therefore , significantly high in U-7 group compared with U-1 group . It seemed necessary to take some counter measure for the induction of TS in the tumor tissue when DB00544 chemotherapy was performed . The optimal dose of DB00515 as a modulator of DB00544 was 1 mg/kg in rat when it was estimated from the changes of intracellular folate levels after administration , which was less than the dose to reveal its own anticancer effect . Glucocorticoid-induced surface expression of annexin 1 blocks beta2-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 ) . 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 . METHODS : Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate ( FP ) , and beta2-integrin adhesion was measured after stimulation with P05113 or eotaxin . Effects of FP on P47712 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 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 beta2-integrin adhesion . Translocation of P47712 to the nuclear membrane was significantly blocked by incubation with FP . Blockade was reversed with annexin 1 blocking antibody . CONCLUSION : Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1 , which blocks P47712 translocation to nuclear membrane . The hyh mutation uncovers roles for alpha Snap in apical protein localization and control of neural cell fate . The hyh ( hydrocephalus with hop gait ) mouse shows a markedly small cerebral cortex at birth and dies postnatally from progressive enlargement of the ventricular system . Here we show that the small hyh cortex reflects altered cell fate . Neural progenitor cells withdraw prematurely from the cell cycle , producing more early-born , deep-layer cerebral cortical neurons but depleting the cortical progenitor pool , such that late-born , upper-layer cortical neurons are underproduced , creating a small cortex. hyh mice carry a hypomorphic missense mutation in the gene Napa encoding soluble N-ethylmaleimide-sensitive factor ( P46459 ) attachment protein alpha ( alpha Snap ) , involved in P60880 receptor ( SNARE ) -mediated vesicle fusion in many cellular contexts . A targeted null Napa mutation is embryonically lethal . Altered neural cell fate is accompanied by abnormal localization of many apical proteins implicated in regulation of neural cell fate , including P12830 , beta-catenin , atypical protein kinase C ( aPKC ) and Q8NI35 ( inactivation-no-afterpotential D-like , also known as protein associated with Lin7 , or Pals1 ) . Apical localization of the SNARE Vamp7 is also disrupted . Thus , alpha Snap is essential for apical protein localization and cell fate determination in neuroepithelial cells . P35367 antagonist cetirizine impairs working memory processing speed , but not episodic memory . BACKGROUND AND PURPOSE : The histaminergic neurotransmitter system is currently under investigation as a target for drug treatment of cognitive deficits in clinical disorders . The therapeutic potential of new drugs may initially be screened using a model of histaminergic dysfunction , for example , as associated with the use of centrally active antihistamines . Of the selective second generation antihistamines , cetirizine has been found to have central nervous system effects . The aim of the present study was to determine whether cetirizine can be used as a tool to model cognitive deficits associated with histaminergic hypofunction . EXPERIMENTAL APPROACH : The study was conducted according to a three-way , double-blind , cross-over design . Treatments were single oral doses of cetirizine 10 and 20 mg and placebo . Effects on cognition were assessed using tests of word learning , memory scanning , vigilance , divided attention , tracking and visual information processing speed . KEY RESULTS : DB00341 10 mg impaired tracking performance and both doses impaired memory scanning speed . None of the other measures indicated impaired performance . CONCLUSION AND IMPLICATIONS : DB00341 affects information processing speed , but these effects were not sufficient to serve as a model for cognitive deficits in clinical disorders . Systems pharmacology assessment of the 5-fluorouracil pathway . AIM : To assess the impact of the 5-fluorouracil ( DB00544 ) drug-pathway genes on cytotoxicity , and determine whether loss-of-function analyses coupled with functional assays can help prioritize pharmacogenomic candidate genes . MATERIALS & METHODS : Dose-response experiments were used to quantify the phenotype of sensitivity to DB00544 following the specific knockdown of genes selected from the DB00544 PharmGKB drug pathway in three human colorectal cell lines . Changes in sensitivity were considered significant if the IC(50) for shRNA-exposed cells were three standard deviations outside the mean IC(50) for control-treated cells . RESULTS : Of the 24 genes analyzed , 13 produced significant changes on the phenotype of sensitivity to DB00544 ( P00374 , Q14117 , P23919 , P33316 , Q05932 , Q92820 , P15531 , Q8TCD5 , P23921 , P04818 , Q9BZX2 , P13051 and P11172 ) . CONCLUSION : The RNAi screening strategy enabled prioritization of the genes from the DB00544 drug pathway . Further validation of the genes credentialed in this study should include gene activity or expression and mutation analyses of clinical samples . DB00368 inhibits exocytosis via the G protein βγ subunit and refilling of the readily releasable granule pool via the α(i1/2) subunit . The molecular mechanisms responsible for the ' distal ' effect by which noradrenaline ( NA ) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in P01308 832/13 β-cells . NA inhibited Ca(2+)-evoked exocytosis by reducing the number of exocytotic events , without modifying vesicle size . Fusion pore properties also were unaffected . NA-induced inhibition of exocytosis was abolished by a high level of Ca(2+) influx , by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ-binding/activating peptide mSIRK . NA-induced inhibition was also abolished by treatment with DB00083 , which cleaves the C-terminal nine amino acids of P60880 , and also by a P60880 C-terminal-blocking peptide containing the DB00083 cleavage site . These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca(2+)](i) and is mediated by an interaction between Gβγ and the C-terminus of P60880 , as is the case for inhibition of neurotransmitter release . Remarkably , in the course of this work , a novel effect of NA was discovered . NA induced a marked retardation of the rate of refilling of the readily releasable pool ( O75783 ) of secretory granules . This retardation was specifically abolished by a Gα(i1/2) blocking peptide demonstrating that the effect is mediated via activation of Gα(i1) and/or Gα(i2) .
[ "DB00341" ]
MH_train_3
MH_train_3
MH_train_3
interacts_with DB00083?
multiple_choice
[ "DB00619", "DB00630", "DB00783", "DB00819", "DB00989", "DB01067", "DB01171", "DB02546", "DB04844" ]
"A protein chip membrane-capture assay for botulinum neurotoxin activity . Botulinum neurotoxins A a(...TRUNCATED)
[ "DB01171" ]
MH_train_4
MH_train_4
MH_train_4
interacts_with DB06813?
multiple_choice
[ "DB00035", "DB00313", "DB00338", "DB00501", "DB00820", "DB00951", "DB01050", "DB02901", "DB08910" ]
"P21554 cannabinoid receptor deficiency promotes cardiac remodeling induced by pressure overload in (...TRUNCATED)
[ "DB01050" ]
MH_train_5
MH_train_5
MH_train_5
interacts_with DB06288?
multiple_choice
[ "DB00035", "DB00290", "DB00293", "DB00338", "DB00341", "DB00951", "DB01200", "DB01296", "DB02901" ]
"Role of monoamine oxidases in the exaggerated 5-hydroxytryptamine-induced tension development of hu(...TRUNCATED)
[ "DB01200" ]
MH_train_6
MH_train_6
MH_train_6
interacts_with DB00862?
multiple_choice
[ "DB00009", "DB00035", "DB00203", "DB00588", "DB00755", "DB00951", "DB02901", "DB04844", "DB06822" ]
"Synthetic delivery system for tuberculosis vaccines : immunological evaluation of the M. tuberculos(...TRUNCATED)
[ "DB00203" ]
MH_train_7
MH_train_7
MH_train_7
interacts_with DB00773?
multiple_choice
[ "DB00290", "DB00452", "DB00630", "DB00783", "DB00977", "DB01067", "DB01656", "DB06779", "DB08910" ]
"Current researches on breast cancer epidemiology in Korea . As a cause of death in women , breast c(...TRUNCATED)
[ "DB01656" ]
MH_train_8
MH_train_8
MH_train_8
interacts_with DB01233?
multiple_choice
[ "DB00015", "DB00341", "DB00741", "DB00820", "DB01576", "DB02546", "DB02901", "DB04844", "DB04905" ]
"Chronic daily tadalafil prevents the corporal fibrosis and veno-occlusive dysfunction that occurs a(...TRUNCATED)
[ "DB04844" ]
MH_train_9
MH_train_9
MH_train_9
interacts_with DB00277?
multiple_choice
[ "DB00338", "DB00351", "DB00459", "DB00588", "DB00820", "DB00989", "DB01182", "DB01259", "DB09068" ]
"Nearly Complete Response of Brain Metastases from P04626 Overexpressing Breast Cancer with DB01259 (...TRUNCATED)
[ "DB01182" ]

Dataset Card for MedHop

With the same format as WikiHop, this dataset is based on research paper abstracts from PubMed, and the queries are about interactions between pairs of drugs. The correct answer has to be inferred by combining information from a chain of reactions of drugs and proteins.

Citation Information

@article{welbl-etal-2018-constructing,
title = Constructing Datasets for Multi-hop Reading Comprehension Across Documents,
author = Welbl, Johannes and Stenetorp, Pontus and Riedel, Sebastian,
journal = Transactions of the Association for Computational Linguistics,
volume = 6,
year = 2018,
address = Cambridge, MA,
publisher = MIT Press,
url = https://aclanthology.org/Q18-1021,
doi = 10.1162/tacl_a_00021,
pages = 287--302,
abstract = {
    Most Reading Comprehension methods limit themselves to queries which
    can be answered using a single sentence, paragraph, or document.
    Enabling models to combine disjoint pieces of textual evidence would
    extend the scope of machine comprehension methods, but currently no
    resources exist to train and test this capability. We propose a novel
    task to encourage the development of models for text understanding
    across multiple documents and to investigate the limits of existing
    methods. In our task, a model learns to seek and combine evidence
    -- effectively performing multihop, alias multi-step, inference.
    We devise a methodology to produce datasets for this task, given a
    collection of query-answer pairs and thematically linked documents.
    Two datasets from different domains are induced, and we identify
    potential pitfalls and devise circumvention strategies. We evaluate
    two previously proposed competitive models and find that one can
    integrate information across documents. However, both models
    struggle to select relevant information; and providing documents
    guaranteed to be relevant greatly improves their performance. While
    the models outperform several strong baselines, their best accuracy
    reaches 54.5 % on an annotated test set, compared to human
    performance at 85.0 %, leaving ample room for improvement.
}
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