interacts_with DB00083?
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 . Pharmacogenetics of antipsychotic-induced weight gain : review and clinical implications . Second-generation antipsychotics ( SGAs ) , such as risperidone , clozapine and olanzapine , are the most common drug treatments for schizophrenia . SGAs presented an advantage over first-generation antipsychotics ( FGAs ) , particularly regarding avoidance of extrapyramidal symptoms . However , most SGAs , and to a lesser degree FGAs , are linked to substantial weight gain . This substantial weight gain is a leading factor in patient non-compliance and poses significant risk of diabetes , lipid abnormalities ( that is , metabolic syndrome ) and cardiovascular events including sudden death . The purpose of this article is to review the advances made in the field of pharmacogenetics of antipsychotic-induced weight gain ( AIWG ) . We included all published association studies in AIWG from December 2006 to date using the Medline and ISI web of knowledge databases . There has been considerable progress reaffirming previous findings and discovery of novel genetic factors . The P28335 and leptin genes are among the most promising , and new evidence suggests that the P14416 , P01375 , P60880 and P32245 genes are also prominent risk factors . Further promising findings have been reported in novel susceptibility genes , such as P21554 , P08183 , ADRA1A and Q9Y5U4 . More research is required before genetically informed , personalized medicine can be applied to antipsychotic treatment ; nevertheless , inroads have been made towards assessing genetic liability and plausible clinical application . Botulinum neurotoxin types A and E require the SNARE motif in P60880 for proteolysis . Botulinum neurotoxins type A and E ( DB00083 and BoNT/E ) are metalloproteases with a unique specificity for P60880 ( synaptosome-associated protein of 25 kDa ) , an essential protein component of the neuroexocytotic machinery . It has been suggested that this specificity is directed through the recognition of a nine residue sequence , termed SNARE motif , that is common to the other two SNARE proteins : VAMP ( vesicle-associated membrane protein ) and syntaxin , the only known substrates of the other six clostridial neurotoxins . Here we analyse the involvement of the four copies of the SNARE motif present in P60880 in its interaction with DB00083 and BoNT/E by following the kinetics of proteolysis of P60880 mutants deleted of SNARE motifs . We show that a single copy of the motif is sufficient for DB00083 and BoNT/E to recognise P60880 . While the copy of the motif proximal to the cleavage site is clearly involved in recognition , in its absence , other more distant copies of the motif are able to support proteolysis . Also , a non-neuronal isoform of P60880 , Syndet , is shown to be sensitive to BoNT/E , but not DB00083 , whilst the P60880 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases . 3,3'-Diindolylmethane induces P55008 arrest and apoptosis in human acute T-cell lymphoblastic leukemia cells . Certain bioactive food components , including indole-3-carbinol ( I3C ) and 3,3'-diindolylmethane ( DIM ) from cruciferous vegetables , have been shown to target cellular pathways regulating carcinogenesis . Previously , our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene ( DBC ) -dependent model of murine T-cell lymphoblastic lymphoma . The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture . Therefore , we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia ( T-ALL ) cells . Treatment of the T-ALL cell lines CCRF-CEM , CCRF-HSB2 , P60880 -T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C . DIM ( 7.5 µM ) arrested CEM and HSB2 cells at the G(1) phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines . In CEM cells , DIM reduced protein expression of cyclin dependent kinases 4 and 6 ( P11802 , Q00534 ) and D-type cyclin 3 ( P30281 ) ; DIM also significantly altered expression of eight transcripts related to human apoptosis ( Q9HD36 , P29965 , O00198 , P01375 , P19438 , Q93038 , P32971 , Q9BUZ4 ) . Similar anticancer effects of DIM were observed in vivo . Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice , reduced final tumor size by 44 % and increased the apoptotic index compared to control-fed mice . Taken together , our results demonstrate a potential for therapeutic application of DIM in T-ALL . Neuritogenic actions of botulinum neurotoxin A on cultured motor neurons . Botulinum neurotoxins ( BoNTs ) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE ) protein cleavage to inhibit neurotransmitter release . The ability of BoNT serotype A ( DB00083 ) to eliminate localized transmitter release at extremely low doses is well characterized . In the current study , we investigated the less understood characteristic of DB00083 to induce nerve outgrowth , sometimes referred to as sprouting . This phenomenon is generally considered a secondary response to the paralytic actions of DB00083 , and other potential factors that may initiate this sprouting have not been investigated . Alternatively , we hypothesized that DB00083 induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor ( SNARE ) synaptosomal associated protein of 25 kDa ( P60880 ) . To test this , the effects of DB00083 application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord . In this system , DB00083 potently stimulated neuritogenesis at concentrations as low as 0.01 nM . The neuritogenic effects of DB00083 exposure were concentration dependent and antagonized by Triticum vulgaris lectin , a known competitive antagonist of BoNT . Similar results were observed with the isolated DB00083 binding domain , revealing that neuritogenesis could be initiated solely by the binding actions of DB00083 . In addition , the presence or absence of P60880 cleavage by DB00083 was not a determinant factor in DB00083 -induced neuritogenesis . Collectively , these results suggest that binding of DB00083 to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s) , independent of its known action on vesicular release . The destructive effect of botulinum neurotoxins on the SNARE protein : P60880 and synaptic membrane fusion . Synaptic exocytosis requires the assembly of syntaxin 1A and P60880 on the plasma membrane and synaptobrevin 2 ( P63027 ) on the vesicular membrane to bridge the two opposite membranes . It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway . The C-terminus of P60880 is known to be the target of botulinum neurotoxins ( DB00083 and BoNT/E ) that block neurotransmitters release in vivo . In this study , we employed electron paramagnetic resonance ( EPR ) spectroscopy to investigate the conformation of the P60880 C-terminus in binary and ternary SNARE complexes . The fluorescence lipid mixing assay shows that the C-terminal of P60880 is essential for membrane fusion , and that the truncated P60880 mutants cleaved by DB00083 and BoNT/E display different inhibition effects on membrane fusion : P60880 -25E ( Δ26 ) abolishes the fusion activity of the SNARE complex , while P60880 -25A ( Δ9 ) loses most of its function , although it can still form a SDS-resistant SNARE complex as the wild-type P60880 . CW-EPR spectra validate the unstable structures of the SNARE complex formed by P60880 mutants . We propose that the truncated P60880 mutants will disrupt the assembly of the SNARE core complex , and then inhibit the synaptic membrane fusion accordingly . A fluorescent ligand-binding alternative using Tag-lite® technology . G-protein-coupled receptors ( GPCRs ) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands . Indeed , 40 % to 50 % of all marketed drugs are thought to modulate GPCR activity , making them the major class of targets in the drug discovery process . Binding assays are widely used to identify high-affinity , selective , and potent GPCR drugs . In this field , the use of radiolabeled ligands has remained so far the gold-standard method . Here the authors report a less hazardous alternative for high-throughput screening ( HTS ) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite(®) . Selective binding of various fluorescent ligands , either peptidic or not , covering a large panel of GPCRs from different classes is illustrated , particularly for chemokine ( P61073 ) , opioid ( δ , µ , and κ ) , and cholecystokinin ( CCK1 and CCK2 ) receptors . Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature . The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing P60880 -tagged GPCR . Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension , in 96- or 384-well plates . Altogether , this new technology offers great advantages in terms of flexibility , rapidity , and user-friendliness ; allows easy miniaturization ; and makes it completely suitable for HTS applications . Alpha-latrotoxin rescues P60880 from DB00083 -mediated proteolysis in embryonic stem cell-derived neurons . The botulinum neurotoxins ( BoNTs ) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex , preventing neurotransmitter release and resulting in neuromuscular paralysis . No pharmacologic therapies have been identified that clinically relieve botulinum poisoning . The black widow spider venom α-latrotoxin ( LTX ) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling . The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons ( ESNs ) , using a novel screening assay designed around the kinetics of DB00083 activation . Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved P60880 within 48 h , whereas treatment with 60 mM K(+) had no effect . Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca(2+) influx and evidence of excitotoxicity , though ESNs remained viable 48 h after LTX treatment . This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity . These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery . They further demonstrate that ESNs are a novel , responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery . DB00877 inhibits in vitro growth and release of angiogenetic factors in human bladder cancer . OBJECTIVES : To evaluate the in vitro effects of rapamycin in human transitional cell carcinoma and to clarify the possible rapamycin-hypoxia interactions . METHODS : Monolayer cultures of RT112 ( P55008 ) , RT4 ( P55008 -2 ) , T24 ( P46379 ) , and P60880 ( G4 ) cells were incubated in medium with or without rapamycin ( 10 microM , 100 microM ) in different gaseous conditions ( 1 % oxygen plus 5 % carbon dioxide plus 94 % nitrogen or 95 % oxygen plus 5 % carbon dioxide or 21 % oxygen plus 78 % nitrogen ) . Cell vitality and proliferation were determined using the microculture tetrazolium assay . Apoptotic cells were quantified by flow cytometry . Enzyme-linked immunosorbent assay was used to visualize inhibition of the mammalian target of rapamycin ( P42345 ) by rapamycin and synthesis of vascular endothelial growth factor . RESULTS : DB00877 delayed proliferation of cancer cell lines but did not induce apoptosis . No direct hypoxia interactions of rapamycin were found . Enzyme-linked immunosorbent assay gave evidence of P42345 inhibition in all cell lines and a significant decrease of hypoxia-induced vascular endothelial growth factor synthesis . CONCLUSIONS : Because proliferation in bladder cancer cells was delayed , but no apoptosis was induced , cell cycle arrest caused by rapamycin was feasible . DB00877 decreased hypoxia-induced synthesis of vascular endothelial growth factor . These findings should be substantiated in an animal model to verify the antiangiogenetic effects of rapamycin . Botulinum neurotoxin A subtype 2 reduces pathological behaviors more effectively than subtype 1 in a rat Parkinson 's disease model . Recent reports indicate that interruption of acetylcholine release by intrastriatal injection of botulinum neurotoxin type A ( DB00083 ) in a rat Parkinson 's disease model reduces pathogenic behavior without adverse side effects such as memory dysfunction . Current knowledge suggests that DB00083 subtype 1 ( BoNT/A1 ) and DB00083 subtype 2 ( BoNT/A2 ) exert different effects . In the present study , we compared the effects of BoNT/A1 and BoNT/A2 on rotation behavior and in vivo cleavage of presynaptic protein P60880 in a rat unilateral 6-hydroxydopamine-induced Parkinson 's disease model . BoNT/A2 more effectively reduced pathogenic behavior by efficiently cleaving P60880 in the striatum compared with that of BoNT/A1 . Our results suggest that BoNT/A2 has greater clinical therapeutic value for treating subjects with Parkinson 's disease compared to that of BoNT/A1 . Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities . A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins . An assay system developed for the detection of botulinum type B neurotoxin ( BoNT/B ) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin , VAMP ( vesicle-associated membrane protein , or synaptobrevin ) . In this assay system , immobilized VAMP ( 60-94 ) peptide substrate is cleaved by BoNT/B at the Gln-76- DB00120 -77 bond , leaving the C-terminal cleavage fragment on the solid phase . This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product . The developed assay was specific to BoNT/B , showing no cross-reactivity with other clostridial neurotoxins , and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml , which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition . Trypsin treatment of BoNT/B samples , which converts the single-chain toxin to the active di-chain form , was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold . An endopeptidase assay for DB00083 , based on the cleavage of a peptide substrate derived from the protein P60880 ( synaptosome-associated protein ) , was also developed and characterized . Quinolinol and peptide inhibitors of zinc protease in botulinum neurotoxin A : effects of zinc ion and peptides on inhibition . Quinolinol derivatives were found to be effective inhibitors of botulinum neurotoxin serotype A ( DB00083 ) . Studies of the inhibition and binding of 7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol ( QAQ ) to the light chain domain ( DB00083 LC ) showed that QAQ is a non-competitive inhibitor for the zinc protease activity . Binding and molecular modeling studies reveal that QAQ binds to a hydrophobic pocket near the active site . Its inhibitor effect does not involve the removal of zinc ion from the light chain . A 24-mer P60880 peptide containing E183 to G206 with Q197C mutation ( Peptide C ) binds to DB00083 LC with an unusually slow second order binding rate constant of 76.7M(-1)s(-1) . QAQ binds to Zn(2+)-free DB00083 LC with a K(D) of 0.67microM and to Peptide C- DB00083 LC complex with a K(D) of 2.33microM . The insights of the interactions of quinolinols and peptides with the zinc protease of DB00083 should aid in the development of inhibitors of metalloproteases . Involvement of the rapamycin-sensitive pathway in the insulin regulation of muscle protein synthesis in streptozotocin-diabetic rats . P01308 resistance in 3-day streptozotocin ( Q11206 ) -treated rats was manifested by the lack of antiproteolytic action of insulin as well as by a reduction of its stimulatory effect on protein synthesis ( -60 % compared with the control group ) in epitrochlearis muscle incubated in vitro . In the present study , we have investigated the diabetes-associated alterations in the insulin signalling cascade , especially the phosphatidylinositol-3 kinase ( P19957 kinase ) / P08133 S6 kinase ( P08133 (S6K) ) pathway , in rat skeletal muscle . LY 294002 , a specific inhibitor of P19957 kinase , markedly decreased the basal rate of protein synthesis and completely prevented insulin-mediated stimulation of this process both in control and diabetic rats . Thus , P19957 kinase is required for insulin-stimulated muscle protein synthesis in diabetic rats as in the controls . DB00877 , an inhibitor of mammalian target of rapamycin ( P42345 ) , had no effect on the basal rate of protein synthesis in either of the experimental groups . In control rats , the stimulatory action of insulin on muscle protein synthesis was diminished by 36 % in the presence of rapamycin , whereas in diabetic muscles this reduction amounted to 68 % . The rapamycin-sensitive pathway makes a relatively greater contribution to the stimulatory effect of insulin on muscle protein synthesis in diabetic rats compared with the controls , due presumably to the preferential decrease in the rapamycin-insensitive component of protein synthesis . Neither basal nor insulin-stimulated P08133 (S6K) activity , a signalling element lying downstream of P42345 , were modified by Q11206 -diabetes . Enhanced production and secretion of glial cell line-derived neurotrophic factor and nerve growth factor from the skin in atopic dermatitis mouse model . Role of neurotrophic factors including nerve growth factor ( P01138 ) in the mechanism of overgrowth and hypersensitivity of sensory nerve in atopic dermatitis ( AD ) has been proposed . P39905 ( P39905 ) is a member of neurotrophic factors of the nervous systems ; however , the role of P39905 in dermatitis is unknown . Q14116 promotes Th2 type allergic condition in skin and various organs in the absence of IL-12 . In this report , we evaluated the expression of P39905 in AD and its association with P01138 and Q14116 . Mice expressing skin-specific Q14116 ( KIL18Tg ) or caspase-1 , an Q14116 converting enzyme , ( KCASP1Tg ) were used as AD models ; P39905 expression was examined by RT-PCR , enzyme immunoassay , and immunohistochemistry . The mRNA expressions of P39905 and P01138 were detected in the epidermis and they were increased in the skin of KIL18Tg and KCASP1Tg mice . P39905 protein production in the skin was also elevated in both transgenic mice and mostly expressed at the basal layer of the epidermis as assessed by immunohistochemistry . Furthermore , the number of nerve fibers was increased in KCASP1Tg , suggesting increased cutaneous innervation . The present results suggest that in addition to P01138 , elevated production and secretion of P39905 in the skin associated with overproduction of Q14116 may also be a potent causative factor of itching in AD . 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 . Concise prediction models of anticancer efficacy of 8 drugs using expression data from 12 selected genes . We developed concise , accurate prediction models of the in vitro activity for 8 anticancer drugs ( DB00544 , DB00515 , DB00305 , DOX , CPT-11 , SN-38 , TXL and TXT ) , along with individual clinical responses to DB00544 using expression data of 12 genes . We first performed cDNA microarray analysis and MTT assay of 19 human cancer cell lines to sort out genes which were correlative in expression levels with cytotoxicities of the 8 drugs ; we selected 13 genes with proven functional significance to drug sensitivity from a huge number of potent prediction marker genes . The correlation significance of each was confirmed using expression data quantified by real-time RT-PCR , and finally 12 genes ( P08183 , Q9UNQ0 , P10632 , P08684 , Q12882 , P09211 , P16455 , P15559 , P16435 , P11388 , P07437 and P04818 ) were selected as more reliable predictors of drug response . Using multiple regression analysis , we fixed 8 prediction formulae which embraced the variable expressions of the 12 genes and arranged them in order , to predict the efficacy of the drugs by referring to the value of Akaike 's information criterion for each sample . These formulae appeared to accurately predict the in vitro efficacy of the drugs . For the first clinical application model , we fixed prediction formulae for individual clinical response to DB00544 in the same way using 41 clinical samples obtained from 30 gastric cancer patients and found to be of predictive value in terms of survival , time to treatment failure and tumor growth . None of the 12 selected genes alone could predict such clinical responses . 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 . Effect of botulinum toxin A on urothelial-release of DB00171 and expression of SNARE targets within the urothelium . AIMS : Botulinum neurotoxin serotype A ( DB00083 ) has emerged as an effective treatment of urinary bladder overactivity . Intravesical lipotoxin ( DB00083 delivery using liposomes ) , which may target the urothelium , is effective in blocking acetic acid induced hyperactivity in animals . The objective of this study was to assess the possible site of toxin action within the urothelium . METHODS : We examined expression of the toxin receptor ( SV2 ) and its cleavage targets ( P60880 and O00161 ) within urothelium as well as effects of the toxin on mechanically evoked release of DB00171 from cultured rat urothelial cells . DB00171 release was measured using the luciferin-luciferase assay ; we examined expression of O00161 and -25 in urothelial cells and mucosa of rat and human bladders . RESULTS : DB00083 ( 1.5 U ; 1-3 hr ) blocked hypotonic evoked release of urothelial DB00171 , without affecting morphology . The expression of protein targets for DB00083 binding ( SV2 ) was detected in human and rat bladder mucosa and catalytic action ( O00161 , -25 ) in urothelial cells and mucosa ( differed in intensity ) from rat and human bladder . Incubation of cultured ( rat ) urothelial cells with DB00083 decreased expression levels of both O00161 ( 44 % ) and P60880 ( 80 % ) . CONCLUSIONS : Our findings reveal that the bladder urothelium expresses the intracellular targets and the binding protein for cellular uptake of DB00083 ; and that the toxin is able to suppress the levels of these targets as well as hypotonic-evoked DB00171 release . These data raise the possibility that intravesical treatment with DB00083 suppresses bladder reflex and sensory mechanisms by affecting a number of urothelial functions including release of transmitters . Structural analysis of botulinum neurotoxin serotype F light chain : implications on substrate binding and inhibitor design . The seven serologically distinct Clostridium botulinum neurotoxins ( BoNTs A-G ) are zinc endopeptidases which block the neurotransmitter release by cleaving one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor complex ( SNARE complex ) essential for the fusion of vesicles containing neurotransmitters with target membranes . These metallopeptidases exhibit unique specificity for the substrates and peptide bonds they cleave . Development of countermeasures and therapeutics for BoNTs is a priority because of their extreme toxicity and potential misuse as biowarfare agents . Though they share sequence homology and structural similarity , the structural information on each one of them is required to understand the mechanism of action of all of them because of their specificity . Unraveling the mechanism will help in the ultimate goal of developing inhibitors as antibotulinum drugs for the toxins . Here , we report the high-resolution structure of active BoNT/F catalytic domain in two crystal forms . The structure was exploited for modeling the substrate binding and identifying the S1 ' subsite and the putative exosites which are different from DB00083 or BoNT/B . The orientation of docking of the substrate at the active site is consistent with the experimental DB00083 -LC: P60880 peptide model and our proposed model for BoNT/E-LC: P60880 . Decreased expression of histamine H1 and H4 receptors suggests disturbance of local regulation in human colorectal tumours by histamine . Production of histamine in colon tumours has been described earlier . DB11320 -mediated signals have been shown to be implicated in tumour growth , and the effects of histamine are largely determined locally by the histamine receptor expression pattern . We analysed histamine receptor expression in human colorectal cancer , adenoma and normal mucosa by quantitative reverse transcription-polymerase chain reaction ( RT-PCR ) , Western blot analysis and immunostaining . Real-time RT-PCR results revealed significantly decreased ( p < 0.001 ) P35367 and Q9H3N8 mRNA levels in tumours compared to normal colonic mucosa , without any significant change in P25021 mRNA expression . Q9Y5N1 was absent in most samples ; it was detected at low levels in 7.9 % of the cases . Protein analysis showed a similar decrease in histamine receptor expression in carcinoma and adenoma compared to normal mucosa controls . Based on these results , we performed further Western blot analysis on Dukes-classified and -selected tumour samples . We found significantly decreased Q9H3N8 levels in neoplastic samples compared to normal colonic tissue , but there was no significant correlation between histamine receptor expression profile and the Dukes stage of tumours . Immunohistochemical staining revealed expression patterns of P35367 , P25021 and Q9H3N8 similar to those suggested by the mRNA and Western blot results . In the present study , we demonstrate that P35367 , P25021 and Q9H3N8 are expressed in colon carcinoma and the adjacent normal mucosa . The results suggest a dramatic alteration in the distribution of histamine receptors in colon cancer . These findings raise the perspective of targeted pharmacological studies with selective histamine receptor antagonists or agonists in the therapy of colorectal tumours . DB01281 inhibits effector T cells through regulatory T cells and TGF-β . The P10747 costimulatory receptor is a critical regulator of T cell function , making it an attractive therapeutic target for the treatment of immune-mediated diseases . DB01281 , now approved for use in humans , prevents naive T cell activation by binding to P33681 proteins and blocking engagement of P10747 . However , DB01281 suppresses inflammation even if administered when disease is established , suggesting alternative mechanisms . We identified a novel , P10747 -independent mechanism by which DB01281 inhibits activated T cells . We show that in vitro , DB01281 synergizes with NO from bone marrow-derived macrophages to inhibit T cell proliferation . Depletion of regulatory T cells ( Tregs ) or interference with TGF-β signaling abrogated the inhibitory effect of DB01281 . Parallel in vivo experiments using an allergic airway inflammation model demonstrated that this novel mechanism required both macrophages and regulatory T cells . Furthermore , DB01281 was ineffective in P84022 -deficient mice , supporting a requirement for TGF-β signaling . Thus , in addition to preventing naive T cells from being fully activated , DB01281 can turn off already activated effector T cells by an NO/regulatory T cell/TGF-β-dependent pathway . This mechanism is similar to cell-extrinsic effects of endogenous P16410 and may be particularly important in the ability of DB01281 to treat chronic inflammatory disease . Cdk5/p35 regulates neurotransmitter release through phosphorylation and downregulation of P/Q-type voltage-dependent calcium channel activity . P12004 -dependent kinase 5 ( Cdk5 ) is a proline-directed serine/threonine kinase with close structural homology to the mitotic Cdks . The complex of Cdk5 and p35 , the neuron-specific regulatory subunit of Cdk5 , plays important roles in brain development , such as neuronal migration and neurite outgrowth . Moreover , Cdk5 is thought to be involved in the promotion of neurodegeneration in Alzheimer 's disease . Cdk5 is abundant in mature neurons ; however , its physiological functions in the adult brain are unknown . Here we show that Cdk5/p35 regulates neurotransmitter release in the presynaptic terminal . Both Cdk5 and p35 were abundant in the synaptosomes . Roscovitine , a specific inhibitor of Cdk5 in neurons , induced neurotransmitter release from the synaptosomes in response to membrane depolarization and enhanced the EPSP slopes in rat hippocampal slices . The electrophysiological study using each specific inhibitor of the voltage-dependent calcium channels ( VDCCs ) and calcium imaging revealed that roscovitine enhanced Ca2+ influx from the P/Q-type VDCC . Moreover , Cdk5/p25 phosphorylated the intracellular loop connecting domains II and III ( L(II-III) ) between amino acid residues 724 and 981 of isoforms cloned from rat brain of the alpha1A subunit of P/Q-type Ca2+ channels . The phosphorylation inhibited the interaction of L(II-III) with P60880 and synaptotagmin I , which were plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein ( P60880 ) receptor ( SNARE ) proteins and were required for efficient neurotransmitter release . These results strongly suggest that Cdk5/p35 inhibits neurotransmitter release through the phosphorylation of P/Q-type VDCC and downregulation of the channel activity . 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 . Steric-dependent label-free and washing-free enzyme amplified protein detection with dual-functional synthetic probes . Enzyme-catalyzed signal amplification with an antibody-enzyme conjugate is commonly employed in many bioanalytical methods to increase assay sensitivity . However , covalent labeling of the enzyme to the antibody , laborious operating procedures , and extensive washing steps are necessary for protein recognition and signal amplification . Herein , we describe a novel label-free and washing-free enzyme-amplified protein detection method by using dual-functional synthetic molecules to impose steric effects upon protein binding . In our approach , protein recognition and signal amplification are modulated by a simple dual-functional synthetic probe which consists of a protein ligand and an inhibitor . In the absence of the target protein , the inhibitor from the dual-functional probe would inhibit the enzyme activity . In contrast , binding of the target protein to the ligand perturbs this enzyme-inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme , thereby activating the enzyme to initiate signal amplification . With this strategy , the fluorescence signal can be amplified to as high as 70-fold . The generality and versatility of this strategy are demonstrated by the rapid , selective , and sensitive detection of four different proteins , avidin , O6-methylguanine DNA methyltransferase ( P16455 ) , P60880 -tag , and lactoferrin , with four different probes . Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant P60880 . Identification of the minimal essential C-terminal residues . Botulinum neurotoxin ( BoNT ) types A and B selectively block exocytosis by cleavage of P60880 and synaptobrevin , respectively ; in humans , many months are required for full recovery from the resultant neuromuscular paralysis . To decipher the molecular basis for such prolonged poisoning , intoxication in adreno-chromaffin cells was monitored over 2 months . Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin . However , inhibition continued in DB00083 -treated cells , throughout the same interval , with a continued predominance of cleaved P60880 -(1-197) over the intact protein . When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length P60880 -(1-206) , no restoration of exocytosis ensued even after 3 weeks . To ascertain if this failure was because of the persistence of the toxin 's protease activity , the cells were transfected with DB00083 -resistant P60880 constructs ; importantly , exocytosis was rescued . C-terminal truncation of the toxin-insensitive P60880 revealed that residues 1-201 , 1-202 , 1-203 afforded a significant return of exocytosis , unlike shorter forms 1-197 , -198 , -199 , or -200 ; accordingly , mutants M202A or L203A of full-length P60880 rescued secretion . These findings give insights into the C-terminal functional domain of P60880 , demonstrate the longevity of DB00083 protease , and provide the prospect of a therapy for botulism . Botulinum neurotoxin A and neurotoxin E cleavage products of synaptosome-associated protein of 25 kd exhibit distinct actions on pancreatic islet beta-cell Kv2.1 channel gating . OBJECTIVES : Synaptosome-associated protein of 25 kd ( P60880 ) regulates pancreatic islet beta-cell-delayed rectifier K channels ( Kv2.1 ) in addition to insulin exocytosis . Botulinum neurotoxin A ( DB00083 ) and E ( BoNT/E ) cleavage and presumed deletion of P60880 have been used to examine P60880 function . We hypothesized that proteolytic products of P60880 ( 206 amino acids ) resulting from DB00083 and BoNT/E cleavage , P60880 (1-197) and P60880 (1-180) , have independent actions on beta-cell Kv gating . METHODS : We examined by confocal microscopy and immunoblotting DB00083 and BoNT/E cleavage of P60880 to these N-terminal fragments , and the consequent effects of these BoNTs and P60880 fragments on Kv currents in rat beta cells and MIN6 cells by patch clamp electrophysiology . RESULTS : Confocal microscopy and immunoblotting showed that MIN6 cells transfected with DB00083 or BoNT/E generated P60880 (1-197) and P60880 (1-180) fragments that were retained in the cytosol . Both BoNTs caused increased rate of channel activation and slowed channel inactivation , mimicked by these P60880 fragments , but not full-length P60880 . These P60880 fragments potentiated tetraethylammonium block of beta-cell Kv currents . CONCLUSIONS : DB00083 or BoNT/E treatment of beta cells generates N-terminal P60880 fragments that are retained in beta cells to directly influence Kv channel gating in a manner distinct from full-length P60880 , contributing to overall actions of these BoNTs on insulin secretion . DB00501 induces interleukin-18 production through H2-agonist activity in monocytes . The present study demonstrates a possible mechanism for the improvement of gastrointestinal cancer patients ' prognosis by the histamine receptor type 2 ( P25021 ) antagonist cimetidine . This agent , but not the P25021 antagonists ranitidine and famotidine , induced the production of an antitumor cytokine , interleukin ( IL ) -18 , by human monocytes and dendritic cells ( DC ) . In fact , ranitidine and famotidine antagonized cimetidine-induced Q14116 production . DB00501 induced the activation of caspase-1 , which is reported to modify immature Q14116 to mature/active Q14116 , and the elevation of intracellular DB02527 , leading to the activation of protein kinase A ( PKA ) . The PKA inhibitor H89 abolished the Q14116 production induced by cimetidine . Moreover , the effects of cimetidine on Q14116 production were reproduced in peripheral blood mononuclear cells from wild-type mice , but not in those from P25021 knockout mice . In conclusion , cimetidine , a partial agonist for P25021 , has a pharmacological profile different from ranitidine and famotidine , possibly contributing to its antitumor activity on gastrointestinal cancers . A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region . Ingestion or inhalation of botulinum neurotoxin ( BoNT ) results in botulism , a severe and frequently fatal disease . Current treatments rely on antitoxins , which , while effective , can not reverse symptoms once BoNT has entered the neuron . For treatments that can reverse intoxication , interest has focused on developing inhibitors of the enzymatic BoNT light chain ( BoNT Lc ) . Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket . To explore the full range of binding sites for serotype A light chain ( DB00083 Lc ) inhibitors , we created a library of non-immune llama single-domain VHH ( camelid heavy-chain variable region derived from heavy-chain-only antibody ) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae . Library selection on DB00083 Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants ( K(d) ) from 230 to 0.03 nM measured by flow cytometry . Eight of 15 VHH inhibited the cleavage of substrate P60880 ( synaptosome-associated protein of 25,000 Da ) by DB00083 Lc . The most potent VHH ( Aa1 ) had a solution K(d) for DB00083 Lc of 1.47 x 10(-)(10) M and an IC(50) ( 50 % inhibitory concentration ) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions . To understand the mechanism by which Aa1 inhibited catalysis , we solved the X-ray crystal structure of the DB00083 Lc-Aa1 VHH complex at 2.6 A resolution . The structure reveals that the Aa1 VHH binds in the alpha-exosite of the DB00083 Lc , far from the active site for catalysis . The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the DB00083 Lc alpha-exosite as a target for inhibitor development . Differential roles of developmentally distinct P60880 isoforms in the neurotransmitter release process . The role of P60880 ( synaptosomal associated protein of 25 kDa ) isotypes in the neurotransmitter release process was examined by varying their relative abundance during PC12 cell differentiation induced by nerve growth factor ( P01138 ) . Norepinephrine release by P01138 -differentiated PC12 cells is more sensitive to type A botulinum toxin ( DB00083 ) than by nondifferentiated cells , while both differentiated and nondifferentiated PC12 cells are equally sensitive to type E botulinum toxin ( BoNT/E ) . The differential sensitivity to DB00083 corresponds to an altered susceptibility of P60880 isotypes to DB00083 cleavage in vitro , whereas both isotypes are equally vulnerable to cleavage by BoNT/E . Using recombinant P60880 preparations , we show that DB00083 cleaves P60880 -25b ( present in differentiated cells ) 2-fold more readily than P60880 -25a ( present in both differentiated and nondifferentiated cells ) . Structural studies using far-ultraviolet circular dichroism ( UV -- CD ) and thermal denaturation suggest a difference in the polypeptide folding as the underlying molecular basis for the differential sensitivity of P60880 -25b and P60880 -25a to DB00083 cleavage . We propose differential roles for P60880 -25b and P60880 -25a in the neurotransmitter release process since our results suggest that DB00083 inhibits neurotransmitter release by primarily cleaving P60880 -25b . 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 . SNARE complex assembly is required for human sperm acrosome reaction . Exocytosis of the acrosome ( the acrosome reaction ) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg . Ca(2+) is an essential mediator of this regulated secretory event . Aided by a streptolysin-O permeabilization protocol developed in our laboratory , we have previously demonstrated requirements for Rab3A , P46459 , and synaptotagmin VI in the human sperm acrosome reaction . Interestingly , Rab3A elicits an exocytotic response of comparable magnitude to that of Ca(2+) . Here , we report a direct role for the SNARE complex in the acrosome reaction . First , the presence of SNARE proteins is demonstrated by Western blot . Second , the Ca(2+)-triggered acrosome reaction is inhibited by botulinum neurotoxins DB00083 , -E , -C , and -F . Third , antibody inhibition studies show a requirement for P60880 , O00161 , syntaxins 1A , 1B , 4 , and 6 , and VAMP 2 . Fourth , addition of bacterially expressed P60880 and O00161 abolishes exocytosis . Acrosome reaction elicited by Rab3-GTP is also inhibited by DB00083 , -C , and -F . Taken together , these results demonstrate a requirement for members of all SNARE protein families in the Ca(2+)- and Rab3A-triggered acrosome reaction . Furthermore , they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes . A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than P60880 substrate . Botulinum neurotoxin serotype A is the most lethal of all known toxins . Here , we report the crystal structure , along with SAR data , of the zinc metalloprotease domain of DB00083 bound to a potent peptidomimetic inhibitor ( K(i)=41 nM ) that resembles the local sequence of the P60880 substrate . Surprisingly , the inhibitor adopts a helical conformation around the cleavage site , in contrast to the extended conformation of the native substrate . The backbone of the inhibitor 's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the " proton shuttle " E224 . This mechanism of inhibition is aided by residue contacts in the conserved S1 ' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets , which are not present in the apo form , especially for the P2 ' residue of the inhibitor . Our inhibitor is specific for DB00083 as it does not inhibit other BoNT serotypes or thermolysin . Characterization of plant P18887 and its interaction with proliferating cell nuclear antigen . In plants , there are no P06746 ( Pol beta ) and P49916 ( Lig3 ) genes . Thus , the plant short-patch base excision repair ( short-patch BER ) pathway must differ considerably from that in mammals . We characterized the rice ( Oryza Sativa L. cv. Nipponbare ) homologue of the mammalian X-ray repair cross complementing 1 ( P18887 ) , a well-known BER protein . The plant P18887 lacks the N-terminal domain ( NTD ) which is required for Pol beta binding and is essential for mammalian cell survival . The recombinant rice P18887 ( OsXRCC1 ) protein binds single-stranded DNA ( ssDNA ) as well as double-stranded DNA ( dsDNA ) and also interacts with rice proliferating cell nuclear antigen ( OsPCNA ) in a pull-down assay . Through immunoprecipitation , we demonstrated that OsXRCC1 forms a complex with P12004 in vivo . OsXRCC1 mRNA was expressed in all rice organs and was induced by application of bleomycin , but not of MMS , H(2)O(2) or UV-B . DB00290 also increased the fraction of OsXRCC1 associated with chromatin . These results suggest that OsXRCC1 contributes to DNA repair pathways that differ from the mammalian BER system . 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 . 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 . Development of a fusion protein SNVP as substrate for assaying multi-serotype botulinum neurotoxins . The SNARE super family has three core members , namely P60880 , P63027 , and syntaxin . P60880 is cleaved by botulinum toxins ( BoNTs ) /A , /C , and /E , whereas P63027 is the substrate for proteolytic BoNTs/B , /D , /F , and /G . In this study , we constructed a hybrid gene encoding the fusion protein SNVP that encompasses P60880 residues Met1 to Gly206 and P63027 residues Met1 to Lys94 . The hybrid gene was cloned in a prokaryotic vector carrying an N-terminal pelB signal sequence and overexpressed in Escherichia coli BL21(DE3) Rosetta . To easily purify the protein , 6× DB00117 double-affinity tags were designed as the linker and C terminus of the fusion protein . SNVP was purified to homogeneity by affinity chromatography on a HisTrap FF column and determined to be more than 97 % pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . N-terminal sequencing of the purified protein showed that signal peptide was successfully removed . The fusion protein SNVP contained the protease cleavage sites of all seven serotypes of BoNTs . SNVP was also proved to be recognized and cleaved by the endopeptidase of BoNTs ( DB00083 -LC , BoNT/B-LC , BoNT/E-LC , and BoNT/G-LC ) . The novel fusion substrate SNVP exhibited high biological activity under the optimal conditions , suggesting its potential use as a reagent for BoNT assay . Expression and purification of the light chain of botulinum neurotoxin A : a single mutation abolishes its cleavage of P60880 and neurotoxicity after reconstitution with the heavy chain . Botulinum neurotoxin type A ( DB00083 ) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings . While the toxin 's heavy ( H ) chain contributes to neuronal binding and internalization , its light ( L ) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa ( P60880 ) . For research and clinical exploitation of this uniquely-acting neurotoxin , recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by DB00135 . The PCR-amplified wild-type and mutant L chain genes were cloned , fused to the gene for maltose-binding protein , and expressed at high levels in Escherichia coli . The soluble fusion proteins were purified using amylose affinity chromatography , and , after factor Xa cleavage , the free L chains were isolated . The wild-type was shown to proteolyze P60880 at a rate approaching that of the native chain while the mutant was inactive . Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo . After reconstitution with the H chain , the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays . This methodology allows , for the first time , routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and , thereby , assist the design of effective inhibitors. ( ABSTRACT TRUNCATED AT 250 WORDS ) The potential role of PD0332991 ( DB09073 ) in the treatment of multiple myeloma . INTRODUCTION : Multiple myeloma ( MM ) remains an incurable malignancy indicating a need for continued investigation of novel therapies . Recent studies have highlighted the role of cyclin-dependent kinases ( CDK ) in the pathogenesis of MM . PD0332991 ( DB09073 ) is an orally bioavailable , highly selective inhibitor of the P11802 /6-cyclin complex and downstream retinoblastoma protein ( Rb ) activation pathway that induces cell cycle arrest in the P55008 phase . AREAS COVERED : In this review , the authors summarize the role of the P11802 /6 signaling pathway in MM . They also summarize the development of PD0332991 as a specific inhibitor of P11802 /6 , and the reported preclinical and clinical data supporting the potential role of PD0332991 in MM . EXPERT OPINION : While PD0332991 is essentially cytostatic , inducing prolonged P55008 arrest , it enhances the cytotoxic effect of other agents effective in MM , including bortezomib and lenalidomide , as confirmed in early phase clinical trials . However , with a plethora of other drugs of different classes being tested in MM , further development of PD0332991 will depend on defining the most efficacious combination with least toxicity . An unexplored opportunity remains the potential protective effect of PD0332991 against lytic bone lesions of MM . The next few years are likely to better define the place of PD0332991 in the treatment of MM . 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 . Glycine insertion at protease cleavage site of P60880 resists cleavage but enhances affinity for botulinum neurotoxin serotype A . The light chain of botulinum neurotoxin A ( DB00083 -LC ) is a Zn-dependent protease that specifically cleaves P60880 of the SNARE complex , thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions . The C-terminus of P60880 ( residues 141-206 ) retains full activity for DB00083 -LC-catalyzed cleavage at P1-P1 ' ( Gln197-Arg198 ) . Using the structure of a complex between the C-terminus of P60880 and DB00083 -LC as a model to design P60880 -derived pseudosubstrate inhibitors ( SNAPIs ) that prevent presentation of the scissile bond to the active site , we introduced multiple DB00117 residues to replace Ala- DB00174 -Gln- DB00125 ( residues 195-198 ) at the substrate cleavage site , with the intent to identify possible side-chain interactions with the active site Zn . We also introduced multiple DB00145 residues between the P1-P1 ' residues to explore the spatial tolerance within the active-site cleft . Using a FRET substrate YsCsY , we compared a series of SNAPIs for inhibition of DB00083 -LC . Among the SNAPIs tested , several known cleavage-resistant , single-point mutants of P60880 were poor inhibitors , with most of the mutants losing binding affinity . Replacement with DB00117 at the active site did not improve inhibition over wildtype substrate . In contrast , DB00145 -insertion mutants were not only resistant to cleavage , but also surprisingly showed enhanced affinity for DB00083 -LC . Two of the DB00145 -insertion mutants exhibited 10-fold lower IC₅₀ values than the wildtype 66-mer P60880 peptide . Our findings illustrate a scenario , where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed . Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system . Botulinum neurotoxins ( BoNTs ) are the most potent natural toxins known . The effects of BoNT serotype A ( DB00083 ) can last several months , whereas the effects of BoNT serotype E ( BoNT/E ) , which shares the same synaptic target , synaptosomal-associated protein 25 ( P60880 ) , last only several weeks . The long-lasting effects or persistence of DB00083 , although desirable for therapeutic applications , presents a challenge for medical treatment of BoNT intoxication . Although the mechanisms for BoNT toxicity are well known , little is known about the mechanisms that govern the persistence of the toxins . We show that the recombinant catalytic light chain ( LC ) of BoNT/E is ubiquitylated and rapidly degraded in cells . In contrast , DB00083 LC is considerably more stable . Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes . In this regard we show that TRAF2 , a RING finger protein implicated in ubiquitylation , selectively associates with BoNT/E LC and promotes its proteasomal degradation . Given these data , we asked whether DB00083 LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains . We describe chimeric P60880 -based ubiquitin ligases that target DB00083 LC for degradation , reducing its duration in a cellular model for toxin persistence . 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) . Regulated secretion is impaired in AtT-20 endocrine cells stably transfected with botulinum neurotoxin type A light chain . Botulinum neurotoxin type A ( DB00083 ) inhibits neurotransmitter release by specific cleavage of P60880 , a synaptosome-associated protein also expressed in the DB01285 secretory cell line AtT-20 . Expression of light chain DB00083 ( L- DB00083 ) gene transfected into AtT-20 cells resulted in a cleaved form of P60880 indistinguishable from that generated by bona fide DB00083 . L- DB00083 -transfected cells showed no difference in replication rate , viability , or phenotype , compared with control AtT-20 cells . In contrast , L- DB00083 -transfected cells could not be induced to secrete DB01285 upon stimulation by 8-bromo- DB02527 or DB00761 . In addition , alpha-latrotoxin induced DB01285 release from control cells , but not from L- DB00083 -transfected cells . These experiments suggest an important role for P60880 in regulated secretion from AtT-20 cells and underline the usefulness of this cell system as a tool for the study of the molecular mechanism of peptide hormone secretion . Truncated P60880 ( 1-197 ) , like botulinum neurotoxin A , can inhibit insulin secretion from HIT-T15 insulinoma cells . We and others have previously shown that insulin-secreting cells of the pancreas express high levels of P60880 ( synaptosomal-associated protein of 25 kDa ) , a 206-amino acid t-SNARE ( target soluble N-ethylmaleimide-sensitive factor attachment protein receptors ) implicated in synaptic vesicle exocytosis . In the present study , we show that P60880 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A ( DB00083 ) into insulin-secreting HIT-T15 cells . Transient expression of DB00083 cleaved the endogenous as well as overexpressed P60880 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin . To determine whether the inhibition of release was due to the depletion of functional P60880 or the accumulation of proteolytic by-products , we transfected cells with P60880 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin . This modified P60880 ( amino acids 1-197 ) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion . Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K(+)-mediated plasma membrane depolarization . Electron microscopy revealed that cells transfected with either DB00083 or truncated P60880 contained significantly higher numbers of insulin granules , many of which clustered close to the plasma membrane . Together , these results demonstrate that functional P60880 proteins are required for insulin secretion and suggest that the inhibitory action of DB00083 toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product , which itself interferes with insulin granule docking and fusion . Excess nitric oxide impairs LXR(α)- O95477 -dependent cholesterol efflux in macrophage foam cells . Excess nitric oxide ( NO ) promotes the progression of atherosclerosis by increasing the oxidation of low-density lipoprotein ( LDL ) and inflammatory responses . However , little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells . In this study , Oil-red O staining , cholesterol and triglyceride assay , Dil-oxidized LDL ( oxLDL ) binding assay , cholesterol efflux assay , real-time RT-PCR and Western blot analysis were used for in vitro experiments . P02649 -deficient ( apoE(-/-) ) and apoE and inducible nitric oxide synthase-deficient ( apoE(-/-) P35228 (-/-) ) mice were as our in vivo models . Treatment with S-nitroso-N-acetyl-D,L-penicillamine ( P60880 ) , an NO donor , exacerbated oxLDL-induced cholesterol accumulation in macrophages , because of reduced efficacy of cholesterol efflux . In addition , P60880 decreased the protein level of DB00171 -binding cassette transporter A1 ( O95477 ) without affecting scavenger receptor type A ( SR-A ) , P16671 , P45844 , or SR-B1 levels . This P60880 -mediated downregulation of O95477 was mainly through the effect of NO but not peroxynitrite . Furthermore , the P60880 -downregulated O95477 was due to the decrease in the liver X receptor α ( LXRα ) -dependent transcriptional regulation . Moreover , genetic deletion of P35228 increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα , O95477 , and Q8WTV0 in aortas and retarded atherosclerosis in apoE(-/-) mice . Our findings provide new insights in the pro-atherogenic effect of excess NO on cholesterol metabolism in macrophages . Induction of nitric oxide synthase in mouse dendritic cells by P01579 , endotoxin , and interaction with allogeneic T cells : nitric oxide production is associated with dendritic cell apoptosis . DB00435 ( NO ) is an important effector molecule that is involved in immune regulation and host defense . In this study , highly purified NLDC 145+ ( O60449 + ) MHC class II(bright) P33681 -2+ dendritic cells ( DC ) propagated from normal mouse bone marrow in response to granulocyte-macrophage P04141 + P05112 were induced to produce NO by P01579 and LPS . NO production was inhibited by the nitric oxide synthase ( NOS ) inhibitor N(G)-monomethyl-L-arginine ( NMMA ) . Nitrite also accumulated in mixed leukocyte culture supernatants as the result of coculture of DC with purified naive allogeneic T cells . Furthermore , NO production was induced by P25942 ligation . Suboptimal T cell proliferation observed at high relative concentrations of DC correlated with increased NO production and was mitigated by NMMA . Induction of mRNA for an inducible NOS ( P35228 ) in DC was confirmed by Northern blotting , whereas intracellular P35228 was visualized by two-color flow cytometry and by both immunofluorescent and immunogold labeling in a subpopulation of P01579 + LPS-stimulated cells . Both endogenous NO production and exposure of unstimulated DC to the NO donor S-nitroso-N-acetyl-penicillamine ( P60880 ) resulted in DC apoptosis . Thus , although DC function initially as the most potent APCs for T cell activation , DC induced to synthesize NOS by P01579 may inhibit ( allogeneic ) T cell proliferation : NO may suppress lymphocyte proliferation and also induce apoptosis of the most potent source of alloantigenic stimulation . DB00501 enhances antigen-specific IgE and Th2 cytokine production . BACKGROUND : Treatment with anti-ulcer drugs has been shown to enhance IgE production against food antigens . However , little is known about the immunological effects of cimetidine , a histamine receptor type 2 ( P25021 ) antagonist that is widely used as an anti-ulcer drug , in allergy . Therefore , the present study investigated the role of cimetidine in Th2 immune responses in mice . METHODS : BALB/c mice were immunized intraperitoneally with ovalbumin ( OVA ) with and without cimetidine . The levels of cytokines in supernatants of spleen cells cultured in the presence of OVA for 4 days and the levels of total and OVA-specific IgG(1) , IgG(2a) and/or IgE in sera from these mice were determined by ELISA . RESULTS : Administration of cimetidine to OVA-sensitized BALB/c mice promoted Th2 cytokine secretion by OVA-stimulated spleen cells in vitro and increased serum levels of OVA-specific IgE , IgG(1) and IgG(2a) . CONCLUSIONS : These results indicate that cimetidine can enhance Th2 responses , suggesting that cimetidine may contribute to IgE production in allergies . 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 . DB00452 -arginine conjugate , a novel HIV-1 Tat antagonist : synthesis and anti-HIV activities . HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease . HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element ( TAR ) RNA , and while secreted extracellularly , it acts as an immunosuppressor , an activator of quiescent T-cells for productive HIV-1 infection , and by binding to CXC chemokine receptor type 4 ( P61073 ) as a chemokine analogue . Here we present a novel HIV-1 Tat antagonist , a neomycin B-hexaarginine conjugate ( NeoR ) , which inhibits Tat transactivation and antagonizes Tat extracellular activities , such as increased viral production , induction of P61073 expression , suppression of CD3-activated proliferation of lymphocytes , and upregulation of the CD8 receptor . Moreover , Tat inhibits binding of fluoresceine isothiocyanate ( FITC ) -labeled NeoR to human peripheral blood mononuclear cells ( PBMC ) , indicating that Tat and NeoR bind to the same cellular target . This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to P61073 . Furthermore , NeoR suppresses HIV-1 binding to cells . Importantly , NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates ( EC(50) = 0.8-5.3 microM ) . A putative model structure for the TAR-NeoR complex , which complies with available experimental data , is presented . We conclude that NeoR is a multitarget HIV-1 inhibitor ; the structure , and molecular modeling and dynamics , suggest its binding to TAR RNA . NeoR inhibits HIV-1 binding to cells , partially by blocking the P61073 HIV-1 coreceptor , and it antagonizes Tat functions . NeoR is therefore an attractive lead compound , capable of interfering with different stages of HIV infection and AIDS pathogenesis . Botulinum neurotoxin type A subtype 2 confers greater safety than subtype 1 in a rat Parkinson 's disease model . Botulinum neurotoxin type A ( DB00083 ) cleaves P60880 and interrupts the release of acetylcholine . We previously reported that DB00083 subtype 2 ( BoNT/A2 ) ameliorates pathologic behavior more effectively than subtype 1 ( BoNT/A1 ) in a rat Parkinson 's disease model . Here , we further show BoNT/A2 has fewer adverse effects than BoNT/A1 . We first confirmed that intrastriatal treatments of both BoNT/As had no-effect on dopaminergic terminals in the striatum . P60880 cleaved by BoNT/A2 was strictly localized to the striatum on the injected side ; however , P60880 cleaved by BoNT/A1 diffused contralaterally . Furthermore , treatment with BoNT/A1 caused a significant reduction in body weight , while BoNT/A2 treatment did not . These results suggest that BoNT/A2 is more beneficial for clinical application against Parkinson 's disease than BoNT/A1 . Distinct kinetic changes in neurotransmitter release after SNARE protein cleavage . Neurotransmitter release is triggered by calcium ions and depends critically on the correct function of three types of SNARE [ soluble N-ethylmaleimide-sensitive factor attachment protein ( P60880 ) receptor ] proteins . With use of the large calyx of Held presynaptic terminal from rats , we found that cleavage of different SNARE proteins by clostridial neurotoxins caused distinct kinetic changes in neurotransmitter release . When elevating calcium ion concentration directly at the presynaptic terminal with the use of caged calcium , cleavage of P60880 by botulinum toxin A ( DB00083 ) produced a strong reduction in the calcium sensitivity for release , whereas cleavage of syntaxin using BoNT/C1 and synaptobrevin using tetanus toxin ( TeNT ) produced an all-or-nothing block without changing the kinetics of remaining vesicles . When stimulating release by calcium influx through channels , a difference between BoNT/C1 and TeNT emerged , which suggests that cleavage of synaptobrevin modifies the coupling between channels and release-competent vesicles . Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active . Botulinum neurotoxins , the most potent of all toxins , induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction . The light chains ( LC ) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins , P60880 , VAMP , or syntaxin at discrete sites . To facilitate the structural and functional characterization of these unique endopeptidases , we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A ( DB00083 ) , overexpressed it in Escherichia coli , and purified the gene product from inclusion bodies . Our procedure can provide 1.1 g of the LC from 1 L of culture . The LC product was stable in solution at 4 degrees C for at least 6 months . This rBoNT/A LC was proteolytically active , specifically cleaving the DB00142 - DB00125 bond in a 17-residue synthetic peptide of P60880 , the reported cleavage site of DB00083 . Its calculated catalytic efficiency kcat/Km was higher than that reported for the native DB00083 dichain . Treating the rBoNT/A LC with mercuric compounds completely abolished its activity , most probably by modifying the cysteine-164 residue located in the vicinity of the active site . About 70 % activity of the LC was restored by adding DB01593 to a DB01593 -free , apo-LC preparation . The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein . In addition , injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing . For the first time , results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form . Evidence for anterograde transport and transcytosis of botulinum neurotoxin A ( DB00083 ) . Botulinum neurotoxin type A ( DB00083 ) is a metalloprotease that blocks synaptic transmission via the cleavage of P60880 ( synaptosomal-associated protein of 25 kDa ) . DB00083 is successfully used in clinical neurology for the treatment of several neuromuscular pathologies and pain syndromes . Despite its widespread use , relatively little is known on DB00083 intracellular trafficking in neurons . Using the visual pathway as a model system , here we show that catalytically active DB00083 is capable of undergoing anterograde axonal transport and transcytosis . Following DB00083 injection into the rat eye , significant levels of DB00083 -cleaved P60880 appeared in the retinorecipient layers of the superior colliculus ( SC ) . Anterograde propagation of DB00083 effects required axonal transport , ruling out a systemic spread of the toxin . Cleaved P60880 was present in presynaptic structures of the tectum , but retinal terminals were devoid of the immunoreactivity , indicative of transcytosis . Experiments based on sequential administration of DB00083 and BoNT/E showed a persistent catalytic activity of DB00083 in tectal cells following its injection into the retina . Our findings demonstrate that catalytically active DB00083 is anterogradely transported from the eye to the SC and transcytosed to tectal synapses . These data are important for a more complete understanding of the mechanisms of action of DB00083 . First report of warfarin dose requirements in patients possessing the P11712 *12 allele . BACKGROUND : DB00682 is the most frequently prescribed anticoagulant in North America and Europe . It is administered as a racemate , but S-warfarin is principally responsible for its anticoagulant activity . Cytochrome P450 ( CYP ) 2C9 is the enzyme primarily responsible for the metabolism of S-warfarin . Numerous variant alleles of P11712 have been identified . The P11712 *12 ( rs9332239 ) allele harbors a P489S substitution in P11712 which has been shown to result in a 40 % decline in catalytic activity in vitro . CASES : Four Caucasian patients with a low mean weekly warfarin dose ( MWWD ) were genotyped for P11712 , Q9BQB6 and P02649 variant alleles . None of the four patients carried the common P11712 variant alleles ( *2 , *3 , *5 , *6 , *7 , *8 , *9 , *11 , *13 ) despite a relatively low MWWD ( 23.4±7.94 mg ) compared to 208 patients carrying the CYP29C9*1 genotype ( 32.2±12.65 mg ) . Given that P11712 *12 confers decreased in vitro activity to the enzyme , we investigated whether these patients carried this allele . All four patients were P11712 *12 CT heterozygotes . Individual comparisons with patients possessing the same Q9BQB6 and P02649 genotypes also demonstrated lower dose requirements in the patients that possessed P11712 *12 allele . CONCLUSIONS : There are no reports of the clinical impact of rs9332239 on P11712 substrates . This is the first report of patients with the rare P11712 *12 genotype and lower warfarin dose requirements . Neuromuscular transmission and muscle contractility in P60880 -deficient coloboma mice . Synaptosomal associated protein of 25 kDa ( P60880 ) is a cytoplasmic protein that participates in the docking and fusion of synaptic vesicles with the nerve terminal in preparation for neurotransmitter release . P60880 is also a substrate for three of the seven serotypes of botulinum neurotoxin ( BoNT ) . Intoxication by DB00083 , /C1 or /E results in weakness and paralysis of skeletal muscle due to cleavage of P60880 ( and syntaxin la in the case /C1 ) at discrete serotype-specific sites . To elucidate the role of P60880 in muscle function in more detail , contractility and neuromuscular transmission were studied in a mutant mouse model termed coloboma . The coloboma mutation results from a contiguous deletion of 1-2 centiMorgans on chromosome 2 , which includes the entire P60880 locus and three other identified genes . Homozygotes do not survive beyond gestation day 6 ; heterozygotes ( Cm/+ ) have a normal life-span but express reduced levels of P60880 mRNA and protein in the brain . The consequences of the Cm/+ mutation on twitch and tetanic tension , quantal release of neurotransmitter and spinal motoneuron expression of P60880 were examined in the present study . Contrary to expectations , Cm/+ mice exhibited no alteration in twitch tension and generated normal tetanic tension even at the highest frequency examined ( 800 Hz ) . Microelectrode recordings revealed that MEPP amplitude and frequency were both within control limits . The ventral spinal cord of Cm/+ mice showed no deficiency in P60880 content and immunohistochemical examination of nerve terminals in Cm/+ mice disclosed that P60880 levels and distribution were similar to those of control mice . It is concluded that spinal motor neurons up-regulate P60880 to preserve vital neuromuscular function . Botulinum neurotoxin A impairs neurotransmission following retrograde transynaptic transport . The widely used botulinum neurotoxin A ( DB00083 ) blocks neurotransmission via cleavage of the synaptic protein P60880 ( synaptosomal-associated protein of 25 kDa ) . Recent evidence demonstrating long-distance propagation of P60880 proteolysis has challenged the idea that DB00083 remains localized to the injection site . However , the extent to which distant neuronal networks are impacted by DB00083 retrograde trafficking remains unknown . Importantly , no studies have addressed whether P60880 cleavage translates into structural and functional changes in distant intoxicated synapses . Here we show that the DB00083 injections into the adult rat optic tectum result in P60880 cleavage in retinal neurons two synapses away from the injection site , such as rod bipolar cells and photoreceptors . Retinal endings displaying cleaved P60880 were enlarged and contained an abnormally high number of synaptic vesicles , indicating impaired exocytosis . Tectal injection of DB00083 in rat pups resulted in appearance of truncated- P60880 in cholinergic amacrine cells . Functional imaging with calcium indicators showed a clear reduction in cholinergic-driven wave activity , demonstrating impairments in neurotransmission . These data provide the first evidence for functional effects of the retrograde trafficking of DB00083 , and open the possibility of using DB00083 fragments as drug delivery vehicles targeting the central nervous system . Development of a fluorescence internal quenching correction factor to correct botulinum neurotoxin type A endopeptidase kinetics using SNAPtide . Botulinum neurotoxins ( BoNTs ) , which are highly toxic proteins responsible for botulism , are produced by different strains of Clostridium botulinum . These various strains of bacteria produce seven distinct serotypes , labeled A-G . Once inside cells , the zinc-dependent proteolytic light chain ( LC ) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis , specifically synaptosomal-associated protein 25 ( P60880 ) for botulinum neurotoxin type A ( DB00083 ) . BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis , detection limits , and cost . SNAPtide , a 13-amino acid fluorescence resonance energy transfer ( FRET ) peptide , was used in this study as a P60880 homologue for the endopeptidase kinetics study of DB00083 LC . SNAPtide uses a fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic acid ( FITC/DABCYL ) FRET pair to produce a signal upon substrate cleavage . Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity , reducing the apparent signal . This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased . In this study , fluorescence internal quenching ( FIQ ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures . The DB00083 LC endopeptidase kinetics at the optimally active temperature ( 37 °C ) using SNAPtide were studied and used to demonstrate the FIQ correction factors in this study . The FIQ correction factors developed provide a convenient method to allow for improved accuracy in determining and comparing DB00083 LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures . Embryonic stem cell-derived neurons are a novel , highly sensitive tissue culture platform for botulinum research . There are no pharmacological treatments to rescue botulinum neurotoxin ( BoNT ) -mediated paralysis of neuromuscular signaling . In part , this failure can be attributed to the lack of a cell culture model system that is neuron-based , allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis , yet still compatible with modern cellular and molecular approaches . We have developed a method to derive highly enriched , glutamatergic neurons from suspension-cultured murine embryonic stem ( ES ) cells . Hypothesizing that ES cell-derived neurons ( ESNs ) might comprise a novel platform to investigate the neurotoxicology of BoNTs , we evaluated the susceptibility of ESNs to DB00083 and BoNT/E using molecular and functional assays . ESNs express neuron-specific proteins , develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions . They express the BoNT substrate SNARE proteins P60880 , P63027 and syntaxin , and treatment with DB00083 and BoNT/E holotoxin results in proteolysis of P60880 within 24 h with EC50s of 0.81 and 68.6 pM , respectively . Intoxication with DB00083 results in the functional inhibition of potassium-induced , calcium-dependent glutamate release . ESNs remain viable and susceptible to intoxication for up to 90 days after plating , enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade . The evidence suggests that derived neurons are a novel , biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line , and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use . 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 . Selective cleavage of SNAREs in sensory neurons unveils protein complexes mediating peptide exocytosis triggered by different stimuli . Oligomerisation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE ) complexes is required for synaptic vesicle fusion and neurotransmitter release . How these regulate the release of pain peptides elicited by different stimuli from sensory neurons has not been established . Herein , K(+) depolarization was found to induce multiple sodium dodecyl sulfate ( SDS ) -resistant SNARE complexes in sensory neurons exposed to botulinum neurotoxins ( BoNTs ) , with molecular weights ranging from 104-288 k ( large ) to 38-104 k ( small ) . Isoform 1 of vesicle-associated membrane protein 1 ( VAMP 1 ) assembled into stable complexes upon depolarisation and was required for the participation of intact synaptosome-associated protein of relative molecular mass 25 k ( P60880 ) or DB00083 -truncated form ( P60880 -25A ) in the large functional and small inactive SDS-resistant SNARE complexes . Cleaving VAMP 1 decreased P60880 -25A in the functional complexes to a much greater extent than the remaining intact P60880 . Syntaxin 1 proved essential for the incorporation of intact and P60880 -25A into the large complexes . Truncation of syntaxin 1 by BoNT/C1 caused /A- and/or /C1-truncated P60880 to appear in non-functional complexes and blocked the release of calcitonin gene-related peptide ( P80511 ) elicited by capsaicin , ionomycin , thapsigargin or K(+) depolarization . Only the latter two were susceptible to /A . Inhibition of P80511 release by DB00083 was reversed by capsaicin and/or ionomycin , an effect overcome by BoNT/C1 . Unlike BoNT/B , BoNT/D cleaved VAMP 1 in addition to 2 and 3 in rat sensory neurons and blocked both P80511 and DB05875 release . Thus , unlike P60880 , syntaxin 1 and VAMP 1 are more suitable targets to abolish functional SNARE complexes and pain peptide release evoked by any stimuli . Catecholamine-related gene expression in blood correlates with tic severity in tourette syndrome . Tourette syndrome ( TS ) is a heritable disorder characterized by tics that are decreased in some patients by treatment with alpha adrenergic agonists and dopamine receptor blockers . Thus , this study examines the relationship between catecholamine gene expression in blood and tic severity . TS diagnosis was confirmed using Diagnostic and Statistical Manual of Mental Disorders ( DSM ) -IV criteria and tic severity measured using the Yale Global Tic Severity Scale ( YGTSS ) for 26 un-medicated subjects with TS . Whole blood was collected and Ribonucleic acid ( RNA ) processed on Affymetrix Human Exon 1.0 ST arrays . An Analysis of Covariance ( ANCOVA ) identified 3627 genes correlated with tic severity ( p < 0.05 ) . Searches of Medical Subject Headings , Gene Ontology , Allen Mouse Brain Atlas , and PubMed determined genes associated with catecholamines and located in the basal ganglia . Using GeneCards , PubMed , and manual curation , seven genes associated with TS were further examined : P14416 , Q9Y5N1 , P27338 , P23560 , P60880 , P31645 , and O75751 . These genes are highly associated with TS and have also been implicated in other movement disorders , Attention Deficit Hyperactivity Disorder ( ADHD ) , and Obsessive-Compulsive Disorder ( OCD ) . Correlation of gene expression in peripheral blood with tic severity may allow inferences about catecholamine pathway dysfunction in TS subjects . Findings built on previous work suggest that at least some genes expressed peripherally are relevant for central nervous system ( CNS ) pathology in the brain of individuals with TS . 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 .